We speculated that iPS cell-reprogramming factors may share common characteristics with pluripotency-associated genes whose perturbed levels in ESCs confer resistance to differentiation. Previous studies have shown that mouse ESCs over-expressing Nanog are resistant to differentiation¹¹, express higher levels of pluripotency-associated genes, and are more effective at reprogramming somatic cells through cell fusion¹². Another transcription factor Tcf3 when depleted in ESCs limits their differentiation ability, and upregulates the expression of pluripotency markers that includes Oct4, Sox2, Nanog and Sall4¹³. As both Nanog and Tcf3 regulate each other, and are core features of the ESC transcriptional network, we speculated that similar to Nanog over-expressing ESCs, the loss of Tcf3 may enhance fusion-mediated reprogramming of somatic hybrid cells. To test this, we used polyethyleneglycol (PEG) to generate duo drug-resistant fusion hybrids between Nanog over-expressing (OE) or Tcf3 RNAi ESCs that were neomycin-resistant (Neo^R) and primary MEFs that were puromycin-resistant (Puro^R) (Figure 1A). Consistent with previous observations, Nanog OE ESCs showed enhanced reprogramming efficiency (Figure 1B & C). Using Tcf3-deficient ESC lines, a significant increase in the number of hybrid clones was also observed (Figure 1B & C). Karyotype analysis confirmed that these were tetraploid (Figure S1). The hybrids possess properties similar to the parental modified ESC lines, including their response to the lack of leukemia inhibitory factor (LIF) and epigenetic reprogramming of the Nanog promoter (Figure S2 & S3). We eliminated the possibility that improvements in reprogramming frequency was attributed to increased cell fusion events¹² (Figure S4).

To dissect the commonalities between Nanog and Tcf3 pathways, we examined the repertoire of genes elevated in Nanog OE and Tcf3 RNAi ESCs that could suggest shared downstream mediators. The intersected expression profiles revealed a handful of genes such as Dazl, Fzd10, Hal, 4930502E18Rik, and Erf that were upregulated in both conditions (Table S1 & S2). Strikingly, Tbx3, a transcription factor previously reported to sustain pluripotency¹⁴ was strongly elevated (Figure 1D; Figure S5A). RNAi knockdown of Tbx3 in ESCs induced differentiation (Figure 1E), with concomitant downregulation of pluripotency-associated genes (Table S3). Tbx3 is also directly bound by both Nanog and Tcf3 (Figure 1F). In reprogrammed ESC/MEF hybrids, Tbx3 levels remained highly elevated (Figure S5B; Table S4 & S5). To test the role of Tbx3 in cell fusion-mediated reprogramming, we fused Neo^R Tbx3 over-expressing ESC lines with MEFs (Figure S6). Indeed, there was an increase in the number of hybrids (Figure 1B & C), that was not due to enhanced cell fusion (Figure S4).

We postulated that Tbx3 may also improve the reprogramming efficiency or the quality of iPS cells. Retroviral infection of MEFs bearing the Oct4-GFP transgene with OSKC induced ~300 ESC-like colonies per 5 × 10⁴ starting cells (Figure 2A). However, only ~10% of these showed activation of the transgene. The addition of Tbx3 (OSKCT) did not increase the frequency of GFP⁺ colony numbers (Figure 2A). With three factors (OSK), the total number of ESC-like colonies was dramatically reduced but 74% of OSK colonies expressed GFP (Figure 2A). The addition of Tbx3 (OSKT) improved the colony count (~38 on average) when compared with OSK (~26), and the percentage of GFP⁺ colonies also significantly increased to 89% (Figure 2A). Other qualitative differences between iPS cells obtained with different factor combinations were observed. OSKC iPS cell colonies were difficult to distinguish amongst transformed and partially reprogrammed cells that did not show GFP expression. OSK and OSKT iPS cell colonies were morphologically similar, with uniform GFP expression within individual colonies (Figure 2B). However, while the activation of Oct4 typically required 14 days post-infection with OSK and OSKC, the use of OSKT took 9-10 days, suggesting that Tbx3 accelerated the reprogramming process (Figure 2C). The efficiency of isolating stable iPS cell lines from GFP⁺ colonies was similar between OSK and OSKT transduction, but almost two-fold higher than OSKC and OSKCT (Figure S7). For all the iPS cell lines obtained, we performed PCR analyses and confirmed genomic integration of the respective transduced genes (Figure S8). Molecular characterization of iPS cells from OSKT confirmed these were alkaline phosphatase-positive, expressed Nanog, Sox2 and SSEA1 (Figure S9). They also form teratomas comprised of multiple differentiated cell types when xenografted into SCID mice (Figure S10).

Next we sought to examine the differences in global transcriptome profiles between iPS cells generated using different factor combinations. To eliminate variations that could have arisen from handling techniques, and to ensure the reproducibility of properties inherent to iPS cell lines generated with different combinations, clones were isolated from two or more independent transduction experiments. Hierarchical clustering revealed that the recently reported iPS cells generated with OS + Esrrb (OSE)⁷ were most dissimilar to wild-type R1 and D3 ESCs with a correlation coefficient (R²) of 0.92 (Figure 3A). Both OSK and OSKT iPS cells bore closer resemblance to ESCs but were indistinguishable from each other; R² = 0.94. Hence, global expression profiling was not sufficiently sensitive in detecting differences between these clones. Closer examination of specific gene level alterations, however, revealed key differences (Figure 3B; Figure S11). We compared the pluripotency-associated gene levels among OSK, OSKT iPS and ES cells. The majority of these genes such as Sall4, Tcf3, Sox2, Zfx, Lin28, Utf1 and Zic3 were non-distinguishing between OSKT and OSK cells as their levels were similar. Surprisingly, a small subset of distinguishing features appeared to define OSKT from OSK cells. Oct4, Nanog, Gdf3, Dppa4 and Tbx3 levels in OSKT cells were equivalent to ESCs, but significantly reduced in OSK cells. This suggests that exogenous Tbx3 may be crucial for assisting in re-establishing proper levels of certain ESC factors critical for the induction of pluripotency that cannot be completely achieved with OSK alone.

We then investigated whether OSKT cells were of higher quality than OSK and OSE cells (Figure S12; summarized in Table S7). We selected numerous iPS cell lines generated from each combination which showed homogeneous activation of Oct4-GFP within each colony: OSKT #1,2,4,6,11-22; OSK #1-3,12-14,16-22; OSE #1,2,9. These lines were derived from four independent transduction experiments performed at different times to eliminate any biasness in clonal selection that may arise from the stochastic behavior of individual clones. Induced-PS cells were then injected into 4-8 cell embryos and cultured in vitro to blastocysts (Figure S13). There was no difference in the maturation efficiency (~95%) between the three iPS cell types (data not shown). All blastocysts transplanted into the surrogate female mice initially had shown robust contribution of GFP⁺ cells to the inner cell mass (ICM) (Figure S13) and live chimeras were obtained. As evaluated by coat color, all OSKT and OSK chimeras showed coats with varying density of black fur, signifying iPS cell contribution; whereas OSE chimeras clearly had lesser black coat (Figure 4C, top panels), indicating OSE cells tend to contribute poorly to chimerism.

More interestingly, obvious differences were discerned between OSKT and OSK cells in their ability to colonize the germ tissues. At E13.5, gonads were obtained from the F₁ chimeric embryos. 34.9% of the gonads from OSKT embryos showed contribution of iPS cells with GFP expression contrasting with 23.6% from OSK and 12.5% from OSE embryos (Figure 4A & B; Figure S14; p<0.01). Further assessment based on GFP distribution within these gonads revealed extensive contribution by OSKT cells as 57.5% of the chimeric gonads were >90% GFP⁺, compared to 26.2% of those from OSK embryos (Figure 4B; p<0.01). This indicates OSKT cells were more efficient in colonizing the germ tissues. Employing one of the most stringent criteria for demonstrating the quality of iPS cell clones, we tested their frequency for germ-line transmission and production of viable F₂ offspring. Once again, we used several iPS cell lines from each combination. Induced-PS cells generated with OSE had very poor capacity for germ-line transmission (Figure 4C, bottom panels; and Figure 4D). Using five chimeras from two lines which were bred with albino mice, a total of seven litters were obtained. Only one litter contained two of 10 offspring that had iPS cell-derived black coat. With OSK, nine chimeras from five lines were used for breeding. 13 litters were produced, of which only two had 100% black offspring, and another four had an average of ~33%. Strikingly, with the nine OSKT chimeras obtained from six lines, nine of 14 litters had 100% black offspring, three had 41%, 50% and 28% each, and only two with none (Figure 4D; p<0.005).

Previous reports had implicated improper retroviral silencing and the frequency of retroviral integrations in altering the efficiency of generating iPS cells and their resultant properties¹⁵. To exclude these possibilities, we confirmed that exogenous expression from the retroviral plasmids was silenced (Figure S15). Southern blot analyses showed that retroviral integration of Oct4, Sox2 and Klf4 transgenes into either OSK or OSKT cells were comparable (Figure S16). Importantly, even with the additional integration of exogenous Tbx3, these cells were consistently of higher quality, thus ruling out the effects of DNA damage. We then speculated that the higher quality OSKT iPS cells could be used to generate viable mice composed entirely of the engineered cells through tetraploid complementation^16,17. This has not been previously shown, perhaps owing to the difficulty of generating high quality iPS cells. Five lines of each OSK and OSKT were tested. In two lines of each, surrogate females were sacrificed at E19 to check for embryos. Only one embryo was obtained out of 66 implanted tetraploid aggregates for OSK cells, whereas four embryos were obtained out of 59 implants for OSKT cells. With the remaining three lines from each, implanted females were followed to term. With OSK lines, only one produced live birth, with a cumulative yield of three births from 105 implants whereas two of the OSKT lines yielded 11 live births out of 107 implants, and two animals continued to thrive beyond ten weeks (Figure 4E).

To better understand how Tbx3 may contribute to improving iPS cell quality, we performed Solexa ChIP-sequencing to uncover the direct regulatory targets of Tbx3 in ESCs (Table S6). Hierarchical clustering of Tbx3 with the previously mapped ESC factors¹⁸ revealed that it shares a large number of common binding sites with the classic pluripotency-associated transcription factors Oct4, Sox2, Nanog and Smad1 (Figure S17). Tbx3 is also found to target ESC factors Oct4, Sox2, Sall4, Lefty1, Lefty2, Zfp42, as well as reprogramming factors Klf2, Klf4, Klf5, n-Myc and c-Myc (Figure S18).

Taken altogether, our study highlights the success of OSKT combination in generating high quality iPS cells capable of germ-line transmission at high efficiency. Previous studies have primarily employed morphological assessments and global gene expression analyses as indicators of iPS cell pluripotency. However, in vitro analyses are not sufficient to distinguish bona fide iPS cells with true ESC properties from poor quality iPS cells which do not possess germ-line competency. The ability of OSKT cells in generating high frequency live birth mice through tetraploid complementation further strengthens the qualitative impact of Tbx3. While Tbx3 retroviral transduction in conjunction with OSK significantly improves iPS cell quality, we noted that not all OSKT clones would manifest robust germ-line transmission. Two reports of more uniform platforms for reprogramming could help address this issue further. Firstly, an inducible system in homogenous starting fibroblast population¹⁹ that includes Tbx3 for reprogramming may allow for derivation and evaluation of iPS clones with more consistent biological properties. Secondly, the dual inhibition of MAPK signaling pathway and GSK3, which greatly enhances the conversion of pre-iPS to fully pluripotent iPS cells⁴ may also lead to the production of iPS clones with more uniform properties.

With the emergence of modified genetic and chemical methods, and an emphasis on the use of minimal reprogramming factors to derive iPS cells, some benchmark as exemplified here by OSKT-derived iPS cells should be employed to evaluate their qualitative and biological properties (Figure S19). The precise mechanistic role for Tbx3 in vastly improving iPS cell quality needs more clarification. The initial ChIP-sequencing data suggests that Tbx3 may be important for the effective re-establishment of the ESC circuitry during the onset of reprogramming, and its subsequent maintenance. The presence of exogenous Tbx3 during the initiation of reprogramming may ensure proper titration of pluripotency-associated and reprogramming factors which are reactivated by OSK to the optimal level. We propose a model whereby the re-establishment of pluripotency from a somatic state is achieved in an increasing probabilistic step-wise manner (Figure S19). The use of different factor combinations results in the generation of iPS populations and clones with markedly varied developmental potentials centered upon attaining progressive “landmarks” of pluripotency. The addition of Tbx3 to a particular combination increases the probabilistic frequency of iPS cells that attain a pluripotent state equivalent or closest to ESCs within the entire population of reprogrammed cells.

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