Maturation, not initiation, is the major roadblock during reprogramming toward pluripotency from human fibroblasts

Koji Tanabe; Michiko Nakamura; Megumi Narita; Kazutoshi Takahashi; Shinya Yamanaka

doi:10.1073/pnas.1310291110

. 2013 Jun 28;110(30):12172–12179. doi: 10.1073/pnas.1310291110

Maturation, not initiation, is the major roadblock during reprogramming toward pluripotency from human fibroblasts

Koji Tanabe ^a, Michiko Nakamura ^a, Megumi Narita ^a, Kazutoshi Takahashi ^a, Shinya Yamanaka ^a,^b,¹

PMCID: PMC3725041 PMID: 23812749

Abstract

Pluripotency can be induced in somatic cells by forced expression of POU domain, class 5, transcription factor 1 (OCT3/4), sex determining region Y-box 2 (SOX2), Kruppel-like factor 4 (KLF4), myelocytomatosis oncogene (c-MYC) (OSKM). However, factor-mediated direct reprogramming is generally regarded as an inefficient and stochastic event. Contrary to this notion, we herein demonstrate that most human adult dermal fibroblasts initiated the reprogramming process on receiving the OSKM transgenes. Within 7 d, ∼20% of these transduced cells became positive for the TRA-1-60 antigen, one of the most specific markers of human pluripotent stem cells. However, only a small portion (∼1%) of these nascent reprogrammed cells resulted in colonies of induced pluripotent stem cells after replating. We found that many of the TRA-1-60–positive cells turned back to be negative again during the subsequent culture. Among the factors that have previously been reported to enhance direct reprogramming, LIN28, but not Nanog homeobox (NANOG), Cyclin D1, or p53 shRNA, significantly inhibited the reversion of reprogramming. These data demonstrate that maturation, and not initiation, is the limiting step during the direct reprogramming of human fibroblasts toward pluripotency and that each proreprogramming factor has a different mode of action.

Keywords: iPS cell, ES cells, HDF, stemness, transcription factor

Induced pluripotent stem cells (iPSCs) were first generated in 2006 by introducing a combination of four transcription factors, POU domain, class 5, transcription factor 1 (OCT3/4), sex determining region Y-box 2 (SOX2), Kruppel-like factor 4 (KLF4), myelocytomatosis oncogene (c-MYC) (OSKM) into embryonic and adult mouse fibroblasts (1). Subsequently, human iPSCs were generated from fibroblasts using either the same factor combination (OSKM) (2) or different, but overlapping, combinations of factors, such as OS plus LIN28 and Nanog homeobox (NANOG) (3). In addition to fibroblasts, iPSCs have been derived from various types of somatic cells, including hepatocytes, gastric epithelial cells (4), blood cells (5), and neural cells (6–8).

Although iPSCs can be reproducibly generated, only a small portion of somatic cells that receive the reprogramming factors become iPSCs. In our initial report (2), ∼10 iPSC colonies emerged from 5 × 10⁵ fibroblasts that were replated 7 d after the transduction of OSKM. This low efficiency (∼0.2%) raised the possibility that the origin of iPSCs is a rare population of stem or progenitor cells that coexists in somatic cell culture. However, this possibility has been formally ruled out, because iPSCs can be generated from terminally differentiated T (9, 10) and B lymphocytes (5) that have undergone genetic recombination. However, the remaining important question is why only a small portion of transduced somatic cells can become iPSCs.

To answer this important question, it is critical to monitor the fate of cells transduced with OSKM during the course of reprogramming, which takes 20–30 d. To this end, it is essential to detect cells that are transduced and are subsequently reprogrammed. In mice, several studies have used a secondary iPSC induction system to uniformly introduce OSKM (6, 11) and specific markers, such as SSEA-1 (12, 13), to detect the cells being reprogrammed. These studies have focused on the molecular events that occur during reprogramming and did not examine the cells that failed to become iPSCs. Furthermore, little is known about the molecular processes of human iPSC generation. In addition, the secondary iPSC induction system may be substantially different from the primary induction by the exogenous delivery of OSKM.

In the current study, we used a SOX2-transgene linked with enhanced GFP (EGFP) with an internal ribosome entry site (IRES) to detect cells that had received the transgenes. We also monitored the cells for the expression of TRA-1-60, a glycoprotein that is expressed in human iPSCs and embryonic stem cells (ESCs), but not in somatic cells. TRA-1-60 is one of the best markers for human pluripotent stem cells (14, 15). By detecting and sorting the EGFP and/or TRA-1-60 (+) cells by flow cytometry, we tried to understand how nascent reprogrammed cells emerge and mature into iPSCs after retroviral transduction of OSKM into human dermal fibroblasts (HDFs) and why most of the transduced cells fail to become iPSCs.

Results

We introduced pMXs retroviral vectors for OCT3/4, KLF4, and c-MYC, together with another pMXs vector carrying SOX2-IRES-EGFP, into 10 HDF lines, which were derived from donors of various ages (0–81 y old), including four Caucasian and six Japanese donors. On day 7 after transduction, ∼20% (5.9–24.5%) of the HDFs became EGFP (+) (Fig. 1 A and B). We sorted the TRA-1-60(+), EGFP (+)/TRA-1-60 (−), and EGFP (−)/TRA-1-60 (−) cells and estimated the copy numbers of the transduced retroviruses by quantitative PCR (qPCR) (Fig. 1A). On day 11 or 15 after transduction, we detected two to five copies per cell of retroviruses for each transgene (OCT3/4, SOX2, KLF4, or c-MYC) in the EGFP (+) cells (Fig. 1C). In contrast, in EGFP (−) cells, we detected one copy or less of each retrovirus. On day 7, we detected approximately twice as many copy numbers of retroviruses in both the EGFP (+) and (−) cells. The reason for the seemingly higher copy numbers on day 7 is unclear. Nevertheless, the results confirmed that EGFP (+) cells represented HDFs that had received higher numbers of retroviral OSKM, whereas EGFP (−) HDFs had integrated significantly fewer copies of retroviral transgenes.

Fig. 1. — Efficiency of iPSC induction. (A) Experimental scheme to analyze the reprogramming process. Magenta dots, TRA-1-60 (+) cells; green dots, transduced cells with SOX2-IRES-EGFP; black dots, HDFs or nontransduced cells. HDFs were introduced with OKM plus SOX2-IRES-EGFP. Transduced cells were replated at day 7 on feeder cells to analyze the generating efficiency of iPSC colonies on day 24. TRA-1-60 (+) cells, EGFP(+)/TRA-1-60 (−) cells, and EGFP(−)/TRA-1-60 (−) cells were sorted using MACS and FACS on days 7, 11, and 15. Sorted TRA-1-60 (+) cells on day 11 were replated on feeder cells to analyze the TRA-1-60 (+) cells on days 20 and 28 and (−) cells on day 20. (B) Proportion of EGFP (+) cells 7 d after OSKM transduction into various HDF lines was analyzed by flow cytometry. The HDFs were derived from various ages (y, year; m, month) of Caucasian and Japanese males (M) and females (F). n = 3. Error bars indicate SD. (C) Number of integrated retroviral transgenes per TRA-1-60 (+), EGFP (+)/TRA-1-60 (−), and EGFP (−)/TRA-1-60 (−) cell on days 7, 11, and 15 was analyzed by a quantitative genomic PCR analysis. Parental nontransduced HDFs were used as a negative control. n = 3. Error bars indicate SD. Numbers of integration in seven established iPSC lines were averaged. (D) Protein levels of OCT3/4, SOX2, KLF4, and c-MYC in TRA-1-60 (+), EGFP (+)/TRA-1-60 (−), and EGFP (−)/TRA-1-60 (−) cells on day 7. (E) Quantification of protein levels in D. Protein levels were adjusted by β-actin for quantification. Protein levels of OCT3/4, SOX2, and c-MYC were normalized with those in HDFs. Protein levels of KLF4 from upper (magenta) and lower (blue) bands are shown separately. Protein levels of KLF4 were normalized with that of upper band in HDFs. n = 3. Error bars indicate SD. (F) Relative RNA expression of transgenous (green) and endogenous (yellow) KLF4 on day 7. All values were normalized with total expression of KLF4 in HDFs. n = 3. Error bars indicate SD. (G) Number of iPSC colonies derived from 2.5 × 10⁵ HDFs transduced with OSKM on day 24. The HDFs were derived from various ages (y, year; m, month) of Caucasian and Japanese males (M) and females (F). n = 3. Error bars indicate SD.

We also examined the protein expression levels of OSKM by Western blot analyses. We found that the protein levels of OCT3/4, SOX2, and c-MYC are similar between TRA-1-60 (+) cells and EGFP (+)/TRA-1-60 (−) cells, being comparable to those in ESCs (Fig. 1 D and E). In contrast, the protein levels of KLF4 in TRA-1-60 (+) cells and EGFP (+)/TRA-1-60 (−) cells were higher than those in ESCs and HDFs. Western blot detected two KLF4 bands. The lower band, which is the major band in ESCs, is more abundant in TRA-1-60 (+) cells than in EGFP (+)/TRA-1-60 (−) cells. However, we did not observe significant differences in KLF4 mRNA levels between TRA-1-60 (+) cells and EGFP (+)/TRA-1-60 (−) cells (Fig. 1F).

On day 7, we replated 2.5 × 10⁵ cells onto STO cells expressing LIF and neomycin-resistant gene (SNL) feeder cells and replaced the fibroblast medium with that for pluripotent stem cells (Fig. 1A). On day 24, we observed 9–583 iPSC colonies (Fig. 1G). Therefore, the putative efficiency of iPSC generation was low, ranging from 0.0036% to 0.23% from replated HDFs, similar to the previously reported results (2).

In contrast to the low efficiency of iPSC generation, we found that ∼20% of EGFP (+) cells became TRA-1-60 (+) on day 7 posttransduction (Fig. 2A). We confirmed that most of the iPSC colonies were derived from these TRA-1-60 (+) cells (Fig. 2B). In contrast, only a small number of iPSC colonies emerged from EGFP (+)/TRA-1-60 (−) cells. We sorted the TRA-1-60 (+) cells on days 7, 11, and 15, and analyzed their gene expression by a microarray analysis. By comparing the gene expression between HDFs and human embryonic stem cells (ESCs), we selected 169 fibroblast-enriched genes (HDF-Gs), of which expression levels are higher at least 100-fold in HDF than in ESCs and 196 ESC-enriched genes (ES-Gs), of which expression levels are higher at least 100-fold in ESCs than in HDFs. We found that approximately half of these ES-Gs were increased at least 10-fold in the TRA-1-60 (+) cells compared with their levels in HDFs on day 7 (Fig. 2C). These included well-known ESC marker genes, such as NANOG, and the endogenous OCT3/4, and their increased expression was confirmed by RT-PCR (Fig. 2D). In contrast, other ESC markers, such as Lin-28 homolog A (LIN28) and endogenous SOX2, remained low. Approximately half of the HDF-Gs decreased by at least 10-fold (Fig. 2C). These data showed that TRA-1-60 (+) cells had acquired a partially reprogrammed state by day 7 posttransduction.

Unexpectedly, we also detected partial reprogramming in the EGFP (+) cells that stayed TRA-1-60 (−) (Fig. 2C). The DNA microarray analyses showed that, of the 196 ES-Gs, the expression of 77 genes increased in EGFP (+)/TRA-1-60 (−) cells by at least 10-fold compared with their levels in HDFs on day 7. Among the 169 HDF-Gs, the expression levels of 53 genes decreased by at least 10-fold. In contrast, the expression of only a small numbers of ES-Gs and HDF-Gs changed >10-fold in the EGFP (−) cells (17 ES-Gs and 2 HDF-Gs). The changes in EGFP (+)/TRA-1-60 (−) cells were similar to, but slightly less prominent than, those in the TRA-1-60 (+) cells (Fig. 2E)

A principal component analysis (PCA) of ES-Gs and HDF-Gs also demonstrated partial reprogramming in TRA-1-60 (+) cells, as well as EGFP (+)/TRA-1-60 (−) cells, but not in EGFP (−) cells (Fig. 2F). Of note, we detected the progression of reprogramming in TRA-1-60 (+) cells on days 7, 11, and 15. In contrast, such progression was not seen in TRA-1-60 (−) cells. These data demonstrated that reprogramming was initiated in the majority of HDFs that had received high copy numbers of the OSKM transgenes but that the maturation of reprogramming only took place in TRA-1-60(+) cells and not in EGFP (+)/TRA-1-60 (−) cells.

We then performed single-cell RT-PCR with Taqman probes that quantitatively detected 13 ES-Gs and 4 HDF-Gs (Fig. 3 A and B; Table S1). In the majority of TRA-1-60 (+) cells on day 7, the expression of nine ES-Gs, including NANOG, LINE-1 type transposase domain containing 1 (L1TD1), growth differentiation factor 3 (GDF3), galanin (GAL), sal-like 4 (SALL4), Apolipoprotein E (APOE), cadherin 1 (CDH1), and Epithelial cell adhesion molecule (EPCAM), increased at least 10-fold from the levels in HDFs. In contrast, the other five ES-Gs, including Developmental pluripotency associated 4 (DPPA4), SOX2, LIN28, DNA methyltransferase 3B (DNMT3B) and gamma-aminobutyric acid (GABA) A receptor, subunit beta 3 (GABRB3), remained suppressed until day 20 or 28. All four HDF-Gs [Matrix metallopeptidase 1 (MMP1), decorin (DCN), Lumican (LUM), and Alanyl (membrane) aminopeptidase (CD13)] were suppressed in the majority of TRA-1-60 (+) cells. The EGFP (+)/TRA-1-60 (−) cells showed similar, but smaller, changes. In sharp contrast, few changes were observed in the expression levels of ES-Gs and HDF-Gs in the EGFP (−) cells. A PCA demonstrated that the reprogramming in TRA-1-60(+) cells gradually progressed from day 7 to 28 (Fig. 3C). The TRA-1-60 (+) cells on days 7, 11, and 15 were more heterogenic in terms of their gene expression than original HDFs or ESCs according to Jensen-Shannon Divergence (JSD) (12) (Fig. 3D). These data confirmed that reprogramming was initiated in most of the HDFs that had received high copy numbers of the OSKM transgenes and that the reprogramming then progressed gradually and specifically in TRA-1-60 (+) cells.

To explore the fate of the nascent reprogrammed cells, we sorted TRA-1-60 (+) cells using magnetic activated cell sorting (MACS) on days 7, 11, 15, and 20 and replated them on SNL feeders. We counted the numbers of iPSC colonies 21 d after seeding (Fig. 4A). The efficiency of iPSC colony formation from TRA-1-60 (+) cells, which were sorted on day 7 or 11, remained low (∼1%). In contrast, the TRA-1-60 (+) cells sorted on day 15 or 20 showed a significantly increased efficiency of iPSC colony formation, indicative of the maturation of reprogramming from day 11 to 15.

Fig. 4. — Reversion during iPSC induction. (A) Generating efficiency of iPSC colonies derived from TRA-1-60 (+) cells on days 7, 11, 15, and 20. TRA-1-60 (+) cells were seeded on feeder cells on each day (day 7, 11, 15, and 20). Numbers of iPSC colonies were counted 21 d after seeding. n = 3. Error bars indicate SD. (B) Proportion of reverted TRA-1-60 (−) cells from TRA-1-60 (+) cells. TRA-1-60 (+) cells were sorted and seeded on feeder cells on different days (days 7, 11, and 15). Proportions of TRA-1-60 (−) cells in the TRA-1-85 (+) cell population were analyzed 4 d after seeding. The human-specific marker, TRA-1-85, was used to distinguish human cells from the mouse feeder cells. n = 3. Error bars indicate SD. (C) PCA of the expression of ES-Gs and HDF-Gs during reprogramming and reversion from the microarray data. Black circles indicate the gene expression patterns of reprogramming cells from HDFs to iPSCs/ESCs. Day 3, EGFP (+) cells. Days 7, 11, 15, and 28: TRA-1-60 (+) cells on each day posttransduction. Colored squares indicate expression patterns of reverted TRA-1-60 (−) cells on days 15 (green) and 20 (magenta) compared with TRA-1-60 (+) cells on day 11. Colored circles indicate nonreverted TRA-1-60 (+) cells on each day.

To further trace the fate of TRA-1-60 (+) cells after sorting, we had to distinguish replated human cells from mouse feeder cells. To this end, we used a human-specific antigen, TRA-1-85. When ESCs or established iPSCs were sorted for TRA-1-60 and were replated, >99% remained positive 4 d after reseeding (Fig. 4B). In contrast, when TRA-1-60 (+) cells were sorted and replated on day 7 after transduction, ∼50% of them reverted and became TRA-1-60 (−) within 4 d after replating. The TRA-1-60 (+) cells sorted on day 11 also showed a strong tendency toward reversion. In contrast, the TRA-1-60 (+) cells sorted on day 15 showed less than 10% reversion (Fig. 4B). Thus, the degree of reversion and the efficiency of iPSC colony formation showed a reverse correlation.

The PCA of 196 ES-Gs and 169 HDF-Gs confirmed that there was a reversion in reprogramming in cells that reverted to TRA-1-60 (−) fate (Figs. 1A and 4C). Compared with TRA-1-60 (+) cells sorted on day 11, cells reverted to negative on days 15 and 20 showed progressive changes in gene expression back to HDFs. In contrast, in the cells that remained TRA-1-60 (+) on days 15 and 20, we detected the progression of reprogramming in the gene expression pattern.

We then investigated the effects of reported proreprogramming factors on various aspects of iPSC generation, including the proliferation of fibroblasts, conversion to TRA-1-60 (+) cells, proliferation of TRA-1-60 (+) cells, death of TRA-1-60 (+) cells, and reversion. We examined the effects of NANOG (3, 16, 17), LIN28 (3), cyclin D1 (18), and p53 shRNA (16, 19–23). We introduced each of these factors, together with OSKM, into HDFs and counted the numbers of iPSC colonies 24 d after transduction. We found that all of these factors, except for NANOG, significantly increased the number of iPSC colonies (Fig. 5A). The proliferation of HDFs was increased by cyclin D1 and p53 shRNA, but not by NANOG or LIN28 (Fig. 5B). The conversion to a TRA-1-60 (+) status was enhanced by LIN28, but not by NANOG, cyclin D1, or p53 shRNA (Fig. 5C). The proliferation of TRA-1-60 (+) cells was also enhanced by LIN28 (Fig. 5D). Thus, the increased conversion may be attributable to the selective expansion of TRA-1-60 (+) cells. The death of TRA-1-60 (+) cells was suppressed by p53 shRNA (Fig. 5E). Reversion from TRA-1-60 (+) to (−) was suppressed by LIN28 (Fig. 5F). In this experiment, LIN28 did not increase total cell number (Fig. 5G). In contrast, p53 shRNA markedly increased both TRA-1-60 (+) and (−) cells. These data demonstrated that each proreprogramming factor has a different mode of action during iPSC generation.

Fig. 5. — Effect of proreprogramming factors on reprogramming. (A) Number of iPSC colonies on day 24 formed from HDFs with OSKM plus different proreprogramming factors. P values were calculated using t tests comparing the different groups to cells with OSKM alone (Mock). All values were normalized to the sample with Mock; n = 3. *P < 0.05. Error bars indicate SD. (B) Proportion of BrdU (+) cells in TRA-1-60 (−) cells on day 11. n = 3. Error bars indicate SD. (C) Relative proportion of TRA-1-60 (+) cells on day 7 posttransduction. All values are normalized to those of cells with OSKM alone (Mock). n = 3. Error bars indicate SD. (D) Proportion of BrdU incorporation into TRA-1-60 (+) cells on day 11. n = 3. Error bars indicate SD. (E) Proportion of apoptotic cells in TRA-1-60 (+) cells on day 11. TRA-1-60 (+) cells were immunostained with Annexin V. n = 3. Error bars indicate SD. (F) Effects of each proreprogramming factor on the reversion of TRA-1-60 (+) to (−) state. Proportion of TRA-1-60 (−) cells in the total population of TRA-1-85 (+) cells 4 d after seeding the TRA1-60 (+) cells, which were sorted on day 11 on feeder cells. n = 3. Error bars indicate SD. (G) Cell number of TRA-1-60 (+) (yellow) and (−) (blue) cells on day 15 from TRA-1-60 (+) cells, which were sorted on day 11. n = 3. Error bars indicate SD.

Discussion

In the current study, we showed that reprogramming was initiated much more frequently than was previously anticipated in human fibroblasts that received the OSKM reprogramming factors. We detected rapid induction of many ES-Gs and suppression of HDF-Gs in the majority of HDFs transduced with high copy numbers of OSKM retroviruses, indicating that reprogramming had been initiated. Approximately 20% of these transduced HDFs became positive for TRA-1-60, one of the best known markers of pluripotent stem cells, within 7 d after transduction. These TRA-1-60 (+) cells showed progressive changes in their gene expression patterns toward those in iPSCs/ESCs. However, only a small portion of TRA-1-60 (+) cells completed the reprogramming process and became iPSCs. Thus, it is maturation, but not initiation, that is responsible for the low efficiency of iPSC generation.

We also showed that one important mechanism underlying the inability of TRA-1-60 (+) cells to complete reprogramming is their reversion to a TRA-1-60 (−) state. When TRA-1-60 (+) cells were sorted and replated on SNL feeder cells on day 7, less than half of them remained positive 4 d after reseeding. Because the proliferation of the reverted TRA-1-60 (−) cells was significantly lower than that of the positive cell (Fig. S1), the actual proportion of cells that reverted to a TRA-1-60 (−) state should be higher than 50%. When cells were sorted on day 11, the reversion rate was still high. In contrast, when they were sorted on day 15, the reversion rate became less than 10%. This result indicates that nascent reprogrammed cells mature during this period (between days 11 and 15).

It remains unclear what distinguishes EGFP (+) cells that become TRA-1-60 (+) from those that remain TRA-1-60 (−) and what distinguishes the TRA-1-60 (+) cells that progress to become iPSCs from those that revert to become TRA-1-60 (−). Of interest, we found that the TRA-1-60 (+) cells on days 7, 11, and 15 were more heterogenic in terms of their gene expression than were both the HDFs and ESCs. It is likely that cells more similar to ESCs in gene expression preferentially progress in the reprogramming process and eventually become iPSCs. However, the reasons for this heterogeneity are also unclear.

It has been reported that the stoichiometry of the four factors affects the formation and quality of iPSCs (24). Although we did not detect significant differences in the retroviral copy numbers between TRA-1-60 (+) cells and TRA-1-60 (−)/EGFP (+) cells, there may be differences in transgene expression due to the integration sites and other mechanisms. Indeed we found that the KLF4 protein level was higher in TRA-1-60 (+) cells than in TRA-1-60 (−)/EGFP (+) cells. Western blot detected two bands of KLF4 and the lower band specifically increased in TRA-1-60 (+) cells. Because we did not observe a significant difference in the KLF mRNA levels between the two types of cell populations, there must be a posttranscriptional regulation of KLF4 transgene that brings the higher protein levels in TRA-1-60 (+) cells. The increased protein level of KLF4 may contribute to the promotion of reprogramming in TRA-1-60 (+) cells.

Another important finding of this study is that each proreprogramming factor has a different mode of action in promoting iPSC generation. We found that three factors, LIN28, cyclin D1, and p53 shRNA, significantly increased the numbers of iPSC colonies when cotransduced with OSKM. However, NANOG failed to show proreprogramming activity in our assay. Among the three factors that did show proreprogramming activities, cyclin D1 and p53 shRNA increased the numbers of iPSC colonies mainly by promoting their proliferation and/or suppressing cell death. In contrast, LIN28 promoted the formation of TRA-1-60 (+) cells and inhibited their conversion back into (−) cells. We found that the endogenous LIN28 was activated later during reprogramming when the TRA-1-60 (+) cells were maturing. Thus, LIN28 seems to promote the maturation of reprogramming. Of note, we found that LIN28 promotes the proliferation of TRA-1-60 (+) cells but not TRA-1-60 (−) cells. This specific activation of nascent reprogrammed cells should contribute to the proreprogramming function of LIN28. Future studies will also be needed to precisely understand the roles of LIN28 in iPSC generation.

In summary, we showed that reprogramming initiated in the majority of HDFs that received sufficient copies of OSKM retroviruses (green dots in Fig. 6). Among these, ∼20% became TRA-1-60 (+) (magenta dots). Reprogramming progressed only in TRA-1-60 (+) cells but not in the EGFP (+)/TRA-1-60 (−) cells. However, most of the TRA-1-60 (+) cells fail to complete reprogramming because of reversion, cell death, and other mechanisms. LIN28 inhibits this reversion, whereas p53 shRNA inhibits cell death and promotes cell proliferation. Thus, it is maturation, and not initiation, that contributes to the low reprogramming efficiency during iPSC generation from HDFs.

Fig. 6. — Model of the reprogramming process. Black dots, HDFs or nontransduced cells; green dots, transduced cells with SOX2-IRES-EGFP; magenta dots, TRA-1-60 (+) cells.

Materials and Methods

Policy on the Statistical Analyses.

All of the quantitative experiments were biologically repeated at least three times. In all figures, the asterisks indicate P < 0.05 as determined by a paired t test. Error bars indicate SDs. The definition of HDF-G and ES-G was a fold-change (FC) > 100, P < 0.05, based on a comparison of 10 independent HDF lines and 10 independent ESC lines (Table S2).

Cell Culture.

HDF lines were purchased from the Japanese Collection of Research Bioresources and Cell Applications. The HDFs were maintained in DMEM (Nacalai) containing 10% FBS (Thermo) (vol/vol) and 0.5% penicillin and streptomycin (Pen/Strep; Invitrogen) (vol/vol). Platinum-E (PLAT-E) cells were cultured in 10% FBS medium with 1 μg/mL puromycin and 10 μg/mL blasticidin S. The ESC lines were obtained from Kyoto University and WiCELL and were maintained in human ESC medium (ReproCELL) supplemented with 4 ng/mL basic FGF (Wako) on mitomycin C–treated SNL feeder cells. All of the cell lines used are listed in Table S2.

Plasmid Construction.

The ORFs of the genes used in this study were amplified by PCR, subcloned into the pENTR-d-TOPO vector (Invitrogen), and verified by sequencing. After that, the ORFs were transferred into the pMXs-gw retroviral vector using a Gateway LR reaction (Invitrogen) according to the manufacturer’s protocol. The knockdown vector for Tumor protein 53 (TP53) was obtained from Addgene (#10672).

iPSC Colony Formation.

Reprogramming was carried out as described in a previous paper (2). To generate retroviruses, we introduced retroviral vectors encoding each factor into PLAT-E cells by using the FuGENE 6 transfection reagent as per the manufacturer’s recommendations. On the following day, we changed the medium to fresh 10% FBS-containing medium and incubated the cells for about 24 h. The medium, including the virus, was then collected and filtered using a 0.45-μm pore size cellulose acetate filter (Whatman). Then, we mixed appropriate combinations of viruses and used them to expose HDFs expressing the mouse Slc7a1 gene overnight with 4 μg/mL polybrene (Nacalai). We designated this point as day 0. Transduced cells were cultured with 10% FBS-containing medium for 7 d. We harvested the cells on day 7 posttransduction and replated 2.5 × 10⁵ cells onto mitomycin C–inactivated SNL feeder cells. The next day, the medium was replaced with human ESC medium. The medium was changed every other day. We counted the number of iPSC colonies on day 24.

Analysis of Reprogrammed and Nonreprogrammed Cells.

The cells transduced with OKM plus SOX2-IRES-EGFP were cultured with 10% FBS-containing medium for 8 d. The culture medium was then replaced with human ESC medium. On days 7, 11, and 15 posttransduction, the transduced cells were harvested using 0.25% trypsin/1 mM EDTA and were filtered using a 70-μm pore size cell strainer (BD Biosciences). The cells were then treated with an anti–TRA-1-60 MicroBead Kit (Miltenyi) and sorted TRA-1-60 (+) cells by an auto-MACS pro device (Miltenyi). EGFP (+)/TRA-1-60 (−) and EGFP (−)/TRA-1-60 (−) cells were sorted by a FACS Aria II instrument (BD Biosciences) from the TRA-1-60 (−) fraction after MACS. To collect the TRA-1-60 (+) cells on days 20 and 28 posttransduction, we replated the MACS-sorted 5 × 10⁵ TRA-1-60 (+) cells on mitomycin C–inactivated SNL feeders in 10-cm culture dishes on day 11 posttransduction. Then, the cells were cultured with Y-27632 (10 μM) in human ESC medium for 2 d. The media were then replaced with fresh human ESC medium every 2 d. On days 20 and 28, the TRA-1-60 (+) cells were sorted using the MACS protocol as described above.

Sorting and Culturing Single TRA-1-60 (+) Cells.

The TRA-1-60 (+) cells were sorted using the MACS protocol as described above. The sorted TRA-1-60 (+) cells were stained with DAPI (Life Technologies) for 30 min to detect the dead cells. Each of the TRA-1-60 (+)/DAPI (−) cells was directly sorted into a well of a 96-well plate on mitomycin C–inactivated SNL feeders using the FACS Aria II instrument. The cells were cultured in human ESC medium with Y-27632 (10 μM). Two days after sorting, we added fresh human ESC medium with Y-27632. We started to replace the medium every 2 d from 4 d after sorting. We counted the number of well in which there were iPSC colonies on day 32.

Flow Cytometry and FACS.

Transduced cells were harvested with 0.25% trypsin/1 mM EDTA on each day after transduction for the analysis. At least 1 × 10⁵ cells were stained with the following antibodies in FACS buffer (2% FBS, 0.36% glucose in PBS) for 30 min at room temperature. The following antibodies were used for the analysis. Alexa 647–conjugated TRA-1-60 (1:20, 560122; BD Biosciences), Alexa 488–conjugated TRA-1-60 (1:20, 560173; BD Biosciences), and phycoerythrin-conjugated TRA-1-85 (1:10, FAB3195P; R&D Systems).

Analysis of Reversion.

We sorted the TRA-1-60 (+) cells using the MACS protocol as described above. The TRA-1-60 (+) cells were cultured with human ESC medium with Y-27632 (10 μM) on mitomycin C–inactivated SNL feeders for 2 d. TRA-1-60 (+) cells were thereafter cultured for either another 2 or 7 d (until day 15 or 20 posttransduction). The media were replaced with fresh human ESC medium every 2 d. To detect the reversion to a TRA-1-60 (−) state, the transduced cells were stained with TRA-1-85 and TRA-1-60 antibodies as previously described in the FACS protocol. The proportion of TRA-1-60 (−) cells in the TRA-1-85 (+) population was calculated to detect the reversion. Reverted TRA-1-60 (−) /TRA-1–85 (+) cells were sorted using the FACS Aria II instrument before the microarray.

Single Cell Gene Expression Analysis.

We first made a 0.2× Taqman probe mix including 1 μL on each 19 Taqman probe (Life Technologies), 4 μL DNA suspension buffer (Tecnova), and 77 μL water. The Taqman probes used in the study are listed up in Table S3. Single cells were directly sorted in 9 μL of master mix (Cells Direct 2× reaction mix; Life Technologies), 5 μL of 0.2× Taqman probe mix, 2.5 μL of Superscript III RT/ Platinum Taq mix (Life Technologies), and 0.2 μL of DNA suspension buffer (Tecnova; 1.3 μL) using the FACS Aria II instrument. The reaction mixture was incubated in a thermal cycler for single cell lysis and reverse transcription at 50 °C for 15 min and for inactivation of reverse transcriptase at 95 °C for 2 min. cDNAs were amplified specifically in TaqMan assays at 95 °C for 15 s and 60 °C for 4 min for 22 cycles. Single-cell qPCR was performed with TaqMan assays, and the amplified cDNAs were diluted by fivefold in 48.48 Dynamic Arrays on a BioMark System (Fluidigm). The Ct values were calculated by the software program provided by the manufacturer (Fluidigm Real-Time PCR Analysis). If Ct values were >26, the expression was filtered out as undetectable/low expression. All TaqMan assays were checked to confirm that they could quantitate the gene expression at the single cell level.

Genomic qPCR.

Genomic DNA was purified using Nucleo spin Tissue XS (Macherey-Nagel). We performed qPCR with SYBR Premix EX Taq II (Takara) and the primers listed in Table S4 to determine the number of integrated transgenes in a genome. FBX15 loci were analyzed as a control to measure the number of whole genome in a reaction. SOX2-IRES-EGFP was detected using primers for EGFP. pMXs vectors encoding each reprogramming factor were used as a reference of qPCR.

qRT-PCR.

RNA samples were purified by miRNeasy kit (Qiagen). The reveres transcription was performed by Rever Tra Ace (Toyobo) at 42 °C for 60 min. We performed qRT-PCR with Universal SYBR green master (Roche) and the primers listed in Table S4.

Western Blot.

Cells were lysed with RIPA buffer (Sigma) as described previously. Twenty micrograms of the cleared lysates was separated on 12% SDS/PAGE. The antibodies used in this study are as follows: anti-OCT3/4 (SC-5279; Santa Cruz), anti-SOX2 (ab97959; Abcam), anti-KLF4 (AF3640; R&D Systems), anti–c-MYC (SC-42; Santa Cruz), anti-mouse IgG-HRP (7076S; Cell Signaling Technology), anti-rabbit IgG-HRP (7074S; Cell Signaling Technology), and anti-goat IgG-HRP (SC-2056; Santa Cruz). The signals were raised and detected by using Immobilon Western (Millipore) and LAS3000 mini (Fuji Film), respectively. The protein levels were quantified using a Multi Gauge software (Fuji Film).

Microarrays.

The total RNA was labeled with Cyanine 3. Samples were hybridized with the Whole Human Genome Microarray SurePrint G3 Human GE 8 × 60K (G4112F; Agilent). Each sample was hybridized once using the one-color protocol. The arrays were scanned with a G2565BA Microarray Scanner System (Agilent). All of the microarray results were analyzed using the GeneSpring v 11 software program (Agilent). Samples were normalized by a 75th percentile shift. Probes were filtered by percentile. If at least one of samples had values within 100 to the 20th percentile, the entities were passed through the filter. Moreover, probes were filtered based on flag values. If the probes had a present or marginal value in at least one of the samples, the probes passed the filter.

BrdU Incorporation.

The medium was changed to fresh media 1 d before the analyses. On the next day, the cells were incubated with 10 μM BrdU for 30 min at 37 °C. Then, the cells were harvested using 0.25% Trypsin/1 mM EDTA and were stained with the anti–TRA1-60 antibody as previously described in the FACS protocol. The BrdU incorporation was detected with a BrdU Flow Kit (BD Pharmingen).

Apoptosis.

The cells transduced with OSKM were harvested on day 11 using 0.25% Trypsin/1 mM EDTA. They were then immediately stained with Allophycocyanin (APC)-conjugated AnnexinV (1:20, 550474; BD Biosciences) in AnnexinV binding buffer. Staining was detected using the FACS Aria II instrument.

Supplementary Material

Supporting Information

supp_110_30_12172__index.html^{(7.2KB, html)}

Acknowledgments

We thank Drs. Hirofumi Suemori and Toshio Kitamura for providing valuable materials; Nikon Corporation for supporting the imaging analyses; Drs. Toshiki Taya, Akira Watanabe, and Takuya Yamamoto for their guidance about the gene expression analyses; Kumiko A. Iwabuchi, Shinsuke Tokumoto, Marie Muramatsu, and Nanako Takizawa for technical assistance; Rie Kato, Eri Minamitani, Sayaka Takeshima, Ryoko Fujiwara, Saki Okamoto, and Yoko Miyake for administrative support; Dr. Eric Rulifson for crucial reading of the manuscript; and the members of the Center for iPS Cell Research and Application for valuable discussions. This study was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), the Ministry of Health, Labor and Welfare, Leading Project of MEXT, and the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program) of the Japanese Society for the Promotion of Science.

Footnotes

Conflict of interest statement: S.Y. is a member without salary of the scientific advisory boards of iPierian, iPS Academia Japan, Megakaryon Corporation, and Retina Institute Japan.

Data deposition: The microarray data analyzed in this paper have been deposited in the Gene Expression Omnibus (GEO) database, http://www.ncbi.nlm.nih.gov/geo/ (accession no. GSE47489).

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1310291110/-/DCSupplemental.

See Profiles on page 9223 in issue 24 of volume 109 and on page 5740 in issue 15 of volume 110.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

supp_110_30_12172__index.html^{(7.2KB, html)}

1310291110_pnas.201310291SI.pdf^{(436.4KB, pdf)}

[r1] 1.Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 2006;126(4):663–676. doi: 10.1016/j.cell.2006.07.024. [DOI] [PubMed] [Google Scholar]

[r2] 2.Takahashi K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 2007;131(5):861–872. doi: 10.1016/j.cell.2007.11.019. [DOI] [PubMed] [Google Scholar]

[r3] 3.Yu J, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science. 2007;318(5858):1917–1920. doi: 10.1126/science.1151526. [DOI] [PubMed] [Google Scholar]

[r4] 4.Aoi T, et al. Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science. 2008;321(5889):699–702. doi: 10.1126/science.1154884. [DOI] [PubMed] [Google Scholar]

[r5] 5.Hanna J, et al. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell. 2008;133(2):250–264. doi: 10.1016/j.cell.2008.03.028. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r6] 6.Wernig M, et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nat Biotechnol. 2008;26(8):916–924. doi: 10.1038/nbt1483. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r7] 7.Eminli S, Utikal J, Arnold K, Jaenisch R, Hochedlinger K. Reprogramming of neural progenitor cells into induced pluripotent stem cells in the absence of exogenous Sox2 expression. Stem Cells. 2008;26(10):2467–2474. doi: 10.1634/stemcells.2008-0317. [DOI] [PubMed] [Google Scholar]

[r8] 8.Kim JB, et al. Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors. Nature. 2008;454(7204):646–650. doi: 10.1038/nature07061. [DOI] [PubMed] [Google Scholar]

[r9] 9.Loh YH, et al. Reprogramming of T cells from human peripheral blood. Cell Stem Cell. 2010;7(1):15–19. doi: 10.1016/j.stem.2010.06.004. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r10] 10.Seki T, et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell. 2010;7(1):11–14. doi: 10.1016/j.stem.2010.06.003. [DOI] [PubMed] [Google Scholar]

[r11] 11.Eminli S, et al. Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nat Genet. 2009;41(9):968–976. doi: 10.1038/ng.428. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r12] 12.Buganim Y, et al. Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell. 2012;150(6):1209–1222. doi: 10.1016/j.cell.2012.08.023. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r13] 13.Polo JM, et al. A molecular roadmap of reprogramming somatic cells into iPS cells. Cell. 2012;151(7):1617–1632. doi: 10.1016/j.cell.2012.11.039. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r14] 14.Chan EM, et al. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat Biotechnol. 2009;27(11):1033–1037. doi: 10.1038/nbt.1580. [DOI] [PubMed] [Google Scholar]

[r15] 15.Andrews PW, Banting G, Damjanov I, Arnaud D, Avner P. Three monoclonal antibodies defining distinct differentiation antigens associated with different high molecular weight polypeptides on the surface of human embryonal carcinoma cells. Hybridoma. 1984;3(4):347–361. doi: 10.1089/hyb.1984.3.347. [DOI] [PubMed] [Google Scholar]

[r16] 16.Hanna J, et al. Direct cell reprogramming is a stochastic process amenable to acceleration. Nature. 2009;462(7273):595–601. doi: 10.1038/nature08592. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r17] 17.Silva J, Chambers I, Pollard S, Smith A. Nanog promotes transfer of pluripotency after cell fusion. Nature. 2006;441(7096):997–1001. doi: 10.1038/nature04914. [DOI] [PubMed] [Google Scholar]

[r18] 18.Edel MJ, et al. Rem2 GTPase maintains survival of human embryonic stem cells as well as enhancing reprogramming by regulating p53 and cyclin D1. Genes Dev. 2010;24(6):561–573. doi: 10.1101/gad.1876710. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r19] 19.Hong H, et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature. 2009;460(7259):1132–1135. doi: 10.1038/nature08235. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r20] 20.Kawamura T, et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature. 2009;460(7259):1140–1144. doi: 10.1038/nature08311. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r21] 21.Utikal J, et al. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature. 2009;460(7259):1145–1148. doi: 10.1038/nature08285. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r22] 22.Marión RM, et al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature. 2009;460(7259):1149–1153. doi: 10.1038/nature08287. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r23] 23.Li H, et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature. 2009;460(7259):1136–1139. doi: 10.1038/nature08290. [DOI] [PMC free article] [PubMed] [Google Scholar]

[r24] 24.Carey BW, et al. Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells. Cell Stem Cell. 2011;9(6):588–598. doi: 10.1016/j.stem.2011.11.003. [DOI] [PubMed] [Google Scholar]

PERMALINK

Maturation, not initiation, is the major roadblock during reprogramming toward pluripotency from human fibroblasts

Koji Tanabe

Michiko Nakamura

Megumi Narita

Kazutoshi Takahashi

Shinya Yamanaka

Series information

Abstract

Results

Fig. 1.

Fig. 2.

Fig. 3.

Fig. 4.

Fig. 5.

Discussion

Fig. 6.

Materials and Methods

Policy on the Statistical Analyses.

Cell Culture.

Plasmid Construction.

iPSC Colony Formation.

Analysis of Reprogrammed and Nonreprogrammed Cells.

Sorting and Culturing Single TRA-1-60 (+) Cells.

Flow Cytometry and FACS.

Analysis of Reversion.

Single Cell Gene Expression Analysis.

Genomic qPCR.

qRT-PCR.

Western Blot.

Microarrays.

BrdU Incorporation.

Apoptosis.

Supplementary Material

Acknowledgments

Footnotes

References

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

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