Cpt1b enhances reprogramming efficiency

To identify candidate genes related to fatty acid metabolism that are involved in the reprogramming process, we analyzed the gene expression profiles of mouse embryonic fibroblasts (MEFs), reprogrammed intermediate phase cells, and iPSCs [21]. A DAVID functional analysis was performed, and the relative expression levels of the candidate genes were confirmed by qRT-PCR and Western blot. The expression of Cpt1b was upregulated during the reprogramming process while Cpt1a was downregulated (Fig. 1a and Additional file 3). To determine the role of Cpt1b in reprogramming, we inserted the coding sequence of Cpt1b into the PiggyBac reprogramming system established by Wang et al. [26]. The efficiency of iPSC induction was increased by overexpressing Cpt1b in combination with the Yamanaka factors during reprogramming (Fig. 1b). To eliminate the potential problem caused by the variable transfection efficiency of the ‘primary’ system, we repeated the experiment using a ‘secondary’ reprogramming system generated by Carey et al. [27]. This system uses MEFs isolated from transgenic mice carrying doxycycline (Dox)-inducible Yamanaka factor constructs. In this system, all MEFs express the reprogramming factors after Dox induction and the reprogrammed cells can be identified by expressing a GFP reporter gene controlled by the Oct4 promoter. In this system, we confirmed higher reprogramming efficiency in response to Cpt1b overexpression. The numbers of alkaline phosphatase (AP)-positive colonies and GFP-positive colonies were both significantly higher in the Cpt1b overexpression group than in the control group (Fig. 1c). Since the ‘secondary’ reprogramming system is more reliable, it was used in the following experiments. Furthermore, the addition of palmitoyl-CoA and carnitine, the substrates for Cpt1b, also increase reprogramming efficiency (see Additional file 4). These results demonstrate that Cpt1b plays an important role in the reprogramming process.

Fig. 1 Cpt1b and palmitoylcarnitine enhance reprogramming efficiency. a Quantitative RT-PCR analysis of cpt1b mRNA levels in mouse embryonic fibroblasts (MEFs) and induced pluripotent stem cells (iPSCs). b Strategy of the PiggyBac Transposon derived reprogramming process and the relative levels of alkaline phosphatase (AP)-positive colonies in the primary reprogramming system with or without cpt1b overexpression. c Strategy of the reprogramming process in the secondary reprogramming system and the relative levels of AP- and green fluorescent protein (GFP)-positive colonies with or without cpt1b overexpression. d Images of AP-stained colonies in control and palmitoylcarnitine (PC)-treated plates. e Relative levels of AP-positive colonies with or without PC (25 μM or 50 μM) in the primary reprogramming system. f Relative levels of AP- and GFP-positive colonies with or without PC (50 μM) in the secondary reprogramming system. Data are presented as the mean ± SEM (n = 3). *P < 0.05; **P < 0.01; ***P < 0.005 (Student’s t test). Cpt carnitine palmitoyltransferase, Dox doxycycline, WT wild-type Full size image

Palmitoylcarnitine, the direct metabolite of the Cpt1b-mediated reaction, enhances reprogramming

Fatty acid metabolism involves three steps: fatty acid activation, mitochondrial transfer of acyl-CoA, and fatty acid oxidation in the mitochondria. Cpt1b is one of the key enzymes involved in the second step, mediating the conversion of palmitoyl-CoA and l-carnitine to l-palmitoylcarnitine. l-Palmitoylcarnitine then passes through the inner membrane of the mitochondria and serves as a primary metabolite for fatty acid oxidation. To confirm whether Cpt1b regulates reprogramming through the modification of fatty acid metabolism, we added palmitoylcarnitine (PC) to the culture medium during the reprogramming process. PC increased the reprogramming efficiency in both reprogramming systems (Fig. 1d–f) with an optimal concentration range of 25 μM to 50 μM (Fig. 1e). PC became lethal at concentrations higher than 50 μM. We also confirmed that PC increased the reprogramming efficiency in the human fibroblast system, indicating a conserved regulatory mechanism (Additional file 5). These results demonstrate that PC, the direct fatty acid metabolite of the Cpt1b-mediated reaction, enhances reprogramming and also suggests that fatty acid oxidation plays an important role in reprogramming.

We compared palmitoylcarnitine-induced pluripotent stem cells (PC-iPSCs) with ESCs and normal iPSCs to confirm that there were no differences in pluripotency. Immunofluorescence indicated no difference in Oct4 or Nanog expression patterns in colonies derived from iPSCs and PC-iPSCs (Fig. 2a). qRT-PCR showed that pluripotency markers were expressed at similar levels in PC-iPSCs compared with those in normal iPSCs and ESCs (Fig. 2b and Additional file 6). Embryonic body (EB) differentiation assays coupled with qRT-PCR showed that markers of all three germ layers were expressed in both cell types (Fig. 2c and Additional file 6). Furthermore, the Pearson correlation coefficient analysis of global gene expression from RNA-seq showed that PC-iPSCs were similar to iPSCs and ESCs (Fig. 2d). The pluripotent gene expression pattern and global gene expression pattern were also similar among PC-iPSCs, iPSCs, and ESCs (Fig. 2e and Additional files 6 and 7). Thus, the above results suggest that PC-iPSCs possess the same degree of pluripotency as normal iPSCs.

Fig. 2 Palmitoylcarnitine induced pluripotent stem cells (PC-iPSCs) have the same pluripotency as embryonic stem cells (ESCs) and iPSCs. a Immunofluorescence staining for pluripotent markers in PC-iPSCs and iPSCs (blue is Hoechst for nucleus, red is Nanog, green is Oct4). Scale bar = 100 μm. b Quantitative RT-PCR analysis of pluripotent markers for ESCs (B6/Blu), iPSCs, and PC-iPSCs. c Quantitative RT-PCR analysis of markers for three germ layers in embryonic bodies (EBs) formed by ESCs, iPSCs, and PC-iPSCs. Gata6: endoderm; FGF5, Otx2: ectoderm; Brachyury, Sox4: mesoderm. d Pearson correlation coefficient analysis of global gene expression in RNA-seq analysis performed on mouse embryonic fibroblasts (MEFs), ESCs, iPSCs, and PC-iPSCs. e Heat map of pluripotent gene expression in RNA-seq analysis performed on MEFs, ESCs, iPSCs, and PC-iPSCs (detailed expression data are shown in Additional file 7). Data are presented as the mean ± SEM (n = 3) Full size image

Fatty acid oxidation regulates reprogramming

To further confirm whether fatty acid oxidation regulates reprogramming, we treated cells with a Cpt1 inhibitor, etomoxir (ETO). ETO blocks the enzymatic activity of Cpt1 family proteins. Treatment with 40 μM ETO markedly inhibited reprogramming (Fig. 3a). This inhibition was rescued by the addition of PC (Fig. 3a), suggesting that the enzymatic activity of Cpt1 family proteins is crucial for reprogramming. This conclusion was confirmed by treating cells with another Cpt1 inhibitor, perhexiline maleate salt (PMS; Additional file 8). The percentage of Oct4-GFP-positive cells, determined by fluorescence-activated cell sorting (FACS), was consistent with the colony counting results (Additional file 8).

Fig. 3 Fatty acid oxidation enhances reprogramming efficiency. a Relative levels of GFP-positive colonies with or without etomoxir (ETO; 40 μM) after reprogramming in the presence of palmitoylcarnitine (PC; 25 μM). b HPLC total ion chromatogram of short-chain acyl-CoA of fatty acid oxidation. c Positive ion electrospray scan mass spectra of acetyl-CoA (810). d Acetyl-CoA (C2-CoA) concentration before and after palmitoylcarnitine treatment. e Multiple reaction monitoring (MRM) chromatograms of long-chain acyl-CoA of fatty acid oxidation. f Lauroyl-CoA (C12-CoA) concentration before and after palmitoylcarnitine treatment. g Myristoyl-CoA (C14-CoA) concentration before and after palmitoylcarnitine treatment. h Relative levels of AP-positive colonies with or without acetylcarnitine (AC; 25 μM) after reprogramming. i Relative levels of AP-positive colonies with or without AC (AC; 25 μM) and perhexiline maleate salt (PMS; 2 μg/ml) after reprogramming. Data are presented as the mean ± SEM (n = 3). *P < 0.05; **P < 0.01, ***P < 0.005 (Student’s t test) Full size image

We also examined the metabolite changes involved in the alteration of fatty acid oxidation by LC-MS. The intracellular level of acetyl-CoA, the final product of fatty acid oxidation, was increased 1 h after PC treatment (Fig. 3b–d and Additional file 9). Furthermore, the levels of lauroyl-CoA and myristoyl-CoA, the direct downstream products of palmitoyl-CoA, were also increased 10 min after PC treatment (Fig. 3e–g and Additional file 9). Interestingly, acetyl-CoA (by acetylcarnitine) also enhanced reprogramming and rescued the PMS/ETO-mediated inhibition of reprogramming, similar to PC (Fig. 3h, i, and Additional file 8). These results confirm that increased fatty acid oxidation promotes reprogramming.

Fatty acid oxidation enhances OXPHOS during early reprogramming

To investigate the functional stage at which fatty acid oxidation affects reprogramming, we measured reprogramming efficiency by treating cells with PC for various times. PC enhanced reprogramming efficiency only at the first week (Fig. 4a) and had no effect when added more than 1 week after Dox induction. The percentage of OCT4-GFP-positive cells determined by FACS was also consistent with the colony counting results (Additional file 8).

Fig. 4 Fatty acid oxidation enhances OXPHOS during early reprogramming. a Relative levels of AP-positive colonies with palmitoylcarnitine (PC) at different stages (1–7 days (7d), 1–14 days (14d), and 1–21 days (21d)) after reprogramming. b Mitostress test of reprogramming mouse embryonic fibroblasts (MEFs) shows the basal oxygen consumption rate (OCR) and the maximal oxidative phosphorylation (OXPHOS) capacity. c Statistical results of maximal OCR levels of (b). d Mitostress test showed the increased maximal OXPHOS capacity after PC treatment (3 days) in normal MEFs and reprogramming MEFs. e Statistical results of maximal OCR levels of (d). f Relative levels of AP-positive colonies with PC in different stages (1–3 days, 1–7 days, and 1–21 days) after reprogramming. Data are presented as the mean ± SEM (n = 3 in a and f, n = 5 in b–e). *P < 0.05 (Student’s t test). Dox doxycycline Full size image

Next, we measured the changes in OXPHOS capacity during induced reprogramming using the Seahorse system. The OXPHOS peak appeared on the third day of reprogramming and subsequently decreased on the fifth day. The OXPHOS capacity was lower in cells after 7 days of reprogramming than in normal MEFs (Fig. 4b, c). The addition of PC in the first 3 days significantly increased the OXPHOS levels in both normal and reprogrammed MEFs, indicating that fatty acid oxidation may help maintain peak OXPHOS capacity, which is important during early reprogramming (Fig. 4d, e). Reprogramming efficiency in the group treated with PC for 3 days was not higher than that in the group treated with PC for 21 days. However, the group treated with PC for 7 days showed the highest efficiency (Fig. 4f). The above results suggest that fatty acid oxidation enhances the early stage of reprogramming by promoting OXPHOS levels, although another pathway that affects reprogramming may also exist.

Protein kinase C is required for fatty acid oxidation function in regulating reprogramming efficiency

To investigate the underlying molecular mechanism, we examined the pathways required for fatty acid oxidation-mediated high reprogramming efficiency. Cell proliferation is one possible target of fatty acid oxidation because increased oxidation may provide more energy and biomaterials for cell division. First, MEFs carrying Dox-inducible Yamanaka factors were cultured with or without Dox at a normal density (2 × 104 cells per well on a six-well plate). PC suppressed the proliferation of MEFs in culture medium without Dox but promoted the proliferation of MEFs when Dox was added (Fig. 5a). When MEFs were cultured at a higher density (1 × 105 cells per well of a six-well plate, a concentration used in the reprogramming experiment) for 48 h, the cell number of the group treated with PC was significantly higher compared with that of the groups without PC treatment. However, after 48 h of Dox induction, PC treatment displayed no effect on cell proliferation (Fig. 5b). The PC-induced increase in cell proliferation may be eliminated by confluent cell interactions. Furthermore, PMS, which blocks reprogramming by inhibiting Cpt1, did not affect cell proliferation (Fig. 5c), suggesting that the enhancement of cell proliferation is not the underlying mechanism by which PC regulates the reprogramming process.

Fig. 5 Palmitoylcarnitine (PC) regulates the phosphorylation of GSK3β in the early reprogramming process via protein kinase C (PKC). a Cell numbers of mouse embryonic fibroblasts (MEFs) cultured in normal or reprogramming media with or without palmitoylcarnitine 3 days after seeding at 2 × 104 cells per well in a six-well plate. b Growth curve of MEFs seeded at 1 × 105 cells per well in a six-well plate in normal or reprogramming media with or without PC. c Growth curve of MEFs seeded at 1 × 105 cells per well in a six-well plate in reprogramming media with or without perhexiline maleate sodium (PMS). d Western blot for phosphorylation change of GSK3β and Erk1/2 at 0, 1, 3, 5, 7, and 9 days after reprogramming. e PKC activity analysis of MEFs with or without PC. f Western blot for the phosphorylation of GSK3β and Erk1/2 in the reprogramming process with or without PC or GF 109203X (GFX) 7 days after reprogramming. g Grayscale analysis of (f). Data are presented as the mean ± SEM (n = 3). *P < 0.05 (Student’s t test). Dox doxycycline Full size image

Although PC is the direct product of Cpt1-mediated fatty acid oxidation, this does not preclude the possibility that PC also affects reprogramming through other pathways. It is known that PC enhances the formation of erythroid colonies [28], suggesting that PC may associate with a hypoxia-like state. To explore this possibility, we measured the protein levels of HIF1α and HIF2α using the primary reprogramming system with and without PC treatment at two different time points in three independent experiments. HIF1α and HIF2α levels were changed only slightly, with no significant differences (Additional file 10).

GSK3β and ERK1/2 are associated with the maintenance of pluripotency and reprogramming. We measured the phosphorylation levels of these two proteins during the early stage of reprogramming. The phosphorylation level of GSK3β was decreased, whereas the phosphorylation of ERK1/2 was increased (Fig. 5d). GSK3β (Ser9) and ERK1/2 are the downstream targets of protein kinase C (PKC), and PC is a well-known PKC inhibitor [29]. We confirmed that PKC activity was inhibited by PC in the secondary reprogramming system (Fig. 5e). Next, we measured the phosphorylation of GSK3β (Ser9) and ERK1/2 in cells cultured with PC and in cells cultured with a PKC inhibitor (GFX). The level of p-GSK3β was reduced following treatment with both PC and GFX. However, the level of p-ERK1/2 was decreased only following GFX treatment (Fig. 5f, g). Moreover, both PKC activity and p-GSK3β were also downregulated in Cpt1b overexpressed cells (Additional file 11). The change in GSK3β phosphorylation occurred during the early stage of reprogramming, which correlated with the observation that PC showed the highest efficiency when introduced within the first 7 days. The decreased p-GSK3β (Ser9) levels suggest increased GSK3β activity [30], which could promote the mesenchymal-to-epithelial transition (MET) process to enhance reprogramming in the early stage, consistent with previous reports [21, 31]. To confirm the function of GSK3β, we treated cells with CHIR99021, an inhibitor of GSK3β, for the first 3 days during iPSC induction. The results showed that inhibition of GSK3β in the early stage decreased reprogramming efficiency (Additional file 12).

Furthermore, the addition of a Cpt1 inhibitor (PMS or ETO) increased the level of p-GSK3β, which was rescued by PC (Fig. 6a, b and Additional file 13). The addition of AC also decreased the phosphorylation level of GSK3β (Fig. 6c, d). These results indicate that fatty acid oxidation is important for PKC activity during the early reprogramming process.

Fig. 6 Fatty acid oxidation-mediated reprogramming efficiency change via downregulation of the protein kinase C (PKC)-GSK3β pathway. a Western blot for the phosphorylation of GSK3β and Erk1/2 in the reprogramming process with or without Cpt1 inhibitors (etomoxir (ETO) or perhexiline maleate salt (PMS)) 3 days after reprogramming. b Grayscale analysis of (a). c Western blot for the phosphorylation of GSK3β and Erk1/2 in the reprogramming process with or without Cpt1 downstream metabolites (palmitoylcarnitine (PC) or acetylcarnitine (AC)) 5 days after reprogramming. d Grayscale analysis of (c). e Relative levels of alkaline phosphatase (AP)-positive colonies with or without GF 109203X (GFX). f Relative levels of AP-positive colonies with GFX in different stages (PC 1–7 days, GFX 1–7 days, and GFX 1–21 days) after reprogramming. g Percentage of Oct4-GFP-positive cells in different stages with different treatments (PC 1–7 days, GFX 1–7 days, and GFX 1–21 days) after reprogramming. FACS plots of green fluorescent protein (GFP) expression are shown. Cutoffs were set using uninduced mouse embryonic fibroblasts (MEFs). The percentages of GFP-positive cells are shown in the right graph. Data are presented as the mean ± SEM (n = 3). *P < 0.05; ***P < 0.005 (Student’s t test). Dox doxycycline Full size image

The addition of GFX (from 1 μM) during the reprogramming process increased reprogramming efficiency (Fig. 6e). Similar to the results obtained following PC treatment, we observed the highest reprogramming efficiency when GFX was introduced during only the first 7 days (Fig. 6f). The percentage of OCT4-GFP-positive cells determined by FACS was also consistent with the colony counting results (Fig. 6g). Together, these results suggest that the function of fatty acid oxidation depends on the activity of PKC.