SCART Journal

Recent advances in reprogramming somatic cells into Induced Pluripotent Stem Cells (iPSCs) provide exciting opportunity for generating renewed tissue for disease modeling and therapy. It has been shown that over-expressing four key transcription factors OCT4 (also known as OCT3/4 or POU5F1), KLF4, SOX2, and c-MYC (known as OKSM) enables reprogramming of mouse and human somatic cells to iPSCs that closely resemble Embryonic Stem Cells(ESCs) [1-6]. However, several issues pertaining to their effectiveness and safety need to be addressed before the technology can be used in clinical settings [1,7]. The major hurdles in the process of developing human cell based therapy include low efficiency, use of retroviral vectors, and tumorigenic potential of the proto-oncogenes c-MYC and KLF4 to generate iPSCs.Moreover, iPSCs generated by various methods vary widely in their differentiation capacityand genomic stability. Therefore, methods to improve the process are being vigorously pursued.

Effortshavebeen made to improve the efficiency of iPSCgeneration[8,9]. Now it is possible to reprogram somatic cells with fewer transcription factors through optimization of methods and use of small molecules. Recent studies have shown that, reprogramming can be achieved by OCT4 over-expression alone in combination with certain small molecules[10,11]. Therefore, OCT4 may serve as a pluripotency determinant in reprogramming. Reprogramming can be facilitated by chromatin-remodeling components of the BAF complex [12] and inhibitors of Histone/Protein Deacetylases(HDACs) and DNA MethylTransferases (DNMTs)[13-17]. Other small molecules like ascorbic acid and GSK3-b inhibitor have also been reported to shorten the process of iPSCs generation [18].Several virus-free methods have been developed to generate iPSCs including using recombinant proteins [19,20], repeated administration of synthetic modified mRNA of five factors (OSKM and LIN28) [21], and episomal plasmids [6,22]. However, their induction efficiencies are still low and may be accompanied by undesirable side effects.

Histone modification and DNA methylation control epigenetic gene expression and are key factors in somatic cell reprogramming. SIRT1 is a member of sirtuins family of NAD⁺-dependent histone/protein deacetylases (class III HDACs)(reviewed in [23]).SIRT1 deacetylates histones, many DNA repair enzymes, DNMT1, the tumor suppressor p53, and other proteins [24-28]. Through this deacetylase activity, SIRT1 modulates gene expression, metabolism, stress response, genomic stability, and longevity [29,30]. Interestingly, SIRT1 expression is higher in stem cells than in differentiated cellsand its levels decline considerably during lineage specification [31-34]. It has been shown that mouse Sirt1 can facilitate iPSCgeneration from Mouse Embryonic Fibroblasts(MEFs) through deacetylatingp53 and Sox2,as well asenhancement of Nanog expression [32,35]. Thus, SIRT1 is important for pluripotency.

OCT4transcription factor promotes self-renewal and inhibits differentiation of stem cells [36,37]. Because ectopic expression of OCT4 alone with small molecules can produce iPSCs[10,11], we initiated a search for factors that can stimulate OCT4 gene expression. In this study, we examined whether SIRT1 can enhance the generation of iPSCs through induction of the pluripotency transcription factors OCT4, KLF4, SOX2, and MYC. We found that SIRT1 stimulates both endogenous and ectopic OCT4 gene expression in human cells.  However, SIRT1 can only stimulate plasmid-derived KLF4, SOX2, and MYC expressions, but not their endogenous genes.Moreover, SIRT1 overexpression induces more iPSC-like colonies.Thus, SIRT1 enhances reprogramming by activating pluripotency genes.

Materials and Methods

Cell Culture

Normal Human Epithelial Keratinocytes (NHEKs, purchased from Lonza) were grown in keratinocyte-SFM medium (Life Technology) with addition of 5 ng/ml epidermal growth factor 1-53 (EGF 1-53), 50 mg/ml Bovine Pituitary Extract (BPE) and 1% penicillin/streptomycin. NHEKs at 3^rd passage were used to generate iPSCs. For iPSCfeeder,Mouse Embryonic Fibroblast (MEF) cells (Millipore)were grown in DMEM (Dulbecco’s Modified Eagle’s Medium, Invitrogen) supplied with 15% Fetal Bovine Serum (FBS), 0.1 mM MEM non-essential amino acids, 2 mM L-glutamine, and 1% penicillin/streptomycinin gelatin-coated dishes to 90% confluence. The cells were then treated with 10 µg/mL mitomycin C (Roche) for 2 hour, washed 3 times with PBS, replaced with fresh medium, and seeded with transfected NHEKs.Sirt1^+/+ (wild-type) and sirt1^−/−(knockout) MEF cells (obtained from Dr. TorenFinkel, National Institutes of Health, Bethesda, MD, U.S.A.) were maintained in DMEM supplemented with 15% FBS and 1% penicillin/streptomycin.

OCT4 Promoter Driven Luciferase Reporter Assay

To facilitate the measurement of OCT4 gene expression, we transfected NHEK cells with OCT4-Luciferase (OCT4-Luc) reporter plasmid (obtained from Dr. Richard Eckert, University of Maryland Medical School, MD, U.S.A.) which contains theOCT4 promoter (2601 nucleotides upstream of transcription start) upstream to the luciferase coding region. Six hours post transfection, histone deacetylaseinhibitors [100 nMtrichostatin A (TSA, Sigma/Aldrich) or 25 mM sirtinol (Sigma/Aldrich)] were added to the cells for 42 hr. To investigate the role of SIRT1 on OCT4 gene expression, we co-transfected SIRT1 expression plasmid (pECE-SIRT1, Addgene) with OCT4-Luc reporter plasmid into NHEK.In addition, Sirt1^+/+ (wild-type) and sirt1^−/−(knockout) MEF cells were transfected with theOCT4-Luc reporter plasmid. Two days post-transfection, cells were collected and subjected for luciferase assay (Invitrogen) according to the manufacturer's protocol.In brief, cells were washed with 1X PBS twice and lysed with lysis reagent.  Cell lysate (20 l) wasmixed with 100 l of luciferase assay reagent and the luciferase activity was measured by a Berthold Lumat LB 9507 Luminometer.

Induced Pluripotent Stem Cells (Ipscs) Generation Using Episomal Vectors

iPSCs were generated from NHEKs by methods as described by Okitaet al.[22] with some modifications (Figure 1).

The three episomalplasmids to express reprogramming factors as described by Okita et al. [22]. CAG, CAG promoter; shRNA, p53 knockdown RNA; WPRE, woodchuck hepatitis post-transcriptional regulatory element; and pA, polyadenylation signal.Schematic of the iPSC induction protocol. Detailed procedures are described in the Materials and Methods section. NHEK: Normal human epithelial keratinocyte; bFGF: basic fibroblast growth factor.

NHEK cells (3^rd passage, 50% confluent) were co-transfected with Okitaepisomal plasmid vectors (pCXLE-hOCT3/4-shp53, pCXLE-hSK, and pCXLE-hUL) [22](referred to as Okita plasmids, Figure 1) with or without pECE-SIRT1. pCXLE-hOCT3/4-shp53 contains OCT4 gene and p53 knockdown RNA (shRNA). pCXLE-hSK contains SOX2and KLF4 genes whilepCXLE-hUL contains L-MYC and LIN28 genes.  These Okita plasmids contain genesencodereprogramming factorsunder the control of CAG promoter which is composed of sequences from the Cytomegalovirus (CMV) early enhancer element; the promoter, the first exon and the first intron of chicken b-actin gene; and the splice acceptor of the rabbit beta-globin gene [38]. Controls are no plasmid and pECEcloningvector.Three days after transfection, a second transfection was performed. Cells were maintained in NHEK medium for 2 weeks before passaging onto MEF feeders in ESC medium (DMEM-F12 with 10% KnockOut™ Serum Replacement, Thermal Fisher), 1% Glutamax(Thermal Fisher), 0.1 mM MEM non-essential amino acids, and8 mg/ml FGF basic (bFGF2) (Life Technology). These cells were maintained and observed for iPSC formation for more than 30 days.  iPSC-like colonies were monitored in a Nikon ELWD 0.3 microscope, counted, and examined live for the presence of the pluripotent cell marker TRA-1-60 (see below). 

Reverse Transcription Quantitative PCR (RT-qPCR)

Total RNA from transfected NHEK cells was isolated by using TRIZOL reagents (Invitrogen) according to the manufacture's protocol. RNA (100 ng) was used as a template for one-step RT-qPCR reactions using primers listed in Table 1.

RT-PCR was performed using the Light Cycler 480 II Detection System (Roche) with iScript™ SYBR® Green One-Step Kit (BioRad, 1708892). The reactions were carried out with an initial step of reverse transcription at 50°C for 30 min and 95^°C for 5 min followed by PCR cycles (95°C for 15 sec and 60°C for 1 min). The mRNA levels of SIRT1 and transcription factors were calculated relative to that of b-actinasDCt which is the difference between the number of cycles required to go above background in respective factors and b-action samples. The fold difference of mRNA levels of Transfected Cells (T) relative to no plasmid control (C) cells was calculated according to the formula 2^{DCt(C)-DCt(T)}.  The reactions were carried out in duplicate and data are averaged from three independenttransfectionexperiments.

ImmunofluorescentStainingwith Stem Cell MarkersOCT4 and TRA-1-60

iPSCs were grown on 12-well culture plate on top of MEF feeders.  Cells were fixed with 4% par formaldehyde at 37^oC for 15 min and permeabilized in 0.25% Triton-X100 at room temperature for 10 minutes. Afterbeing washed with PBS and incubatedin blocking buffer (0.5% NP-40 and 5% bovine serum albumin in PBS) for 30 min, the cells wereincubated with anti-OCT3/4 antibody(Santa Cruz Biotechnology) in blocking buffer at 4^oCovernight. Next, the cells were washed three times with PBS and stained with Alexa Fluor 488goat anti-mouse IgG antibody (Invitrogen) and 5 mg/ml Alexa Fluor 594 anti-human TRA-1-60-R antibody (Biolegend) for 2 hour. After being washed three times with PBS, nuclear DNAwascounterstained with 500 nMDAPI(4’,6’-diamidino-2-phenyllindole, Vector Laboratory) in PBS for 10 minute.Images were captured with a Nikon PCM 2000 confocal microscope scanning system.

Results

SirtinolsuppressesandSIRT1 Enhances OCT4 Expression.

Reprogramming somatic cells into iPSCs erases somatic epigenetic signatures (DNA methylation and histone modification) and establishes alternative epigenetic marks of ESCs[40]. Histone acetylation induces an open chromatin configuration associated with gene activation[41]. The level of histone acetylation is controlled by Histone/Protein Acetyltransferases(HATs) and Histone/Protein Deacetylases(HDACs) [42,43]. To test whether HDACs regulate iPSC generation from NHEK cells, we first tested whether their inhibition regulatesOCT4 expression. We employed two HDAC inhibitors: TrichostatinA (TSA), a pan inhibitor targeting class I/II HDACs and sirtinol, a pan inhibitor for class III HDACs [43]. We transfected NHEK cells with the OCT4-Luc reporter plasmid whichcontainstheOCT4 promoter upstream to the luciferase coding region to facilitate the measurement of OCT4 gene expression.Six hours post transfection,wetreated the cell with the HDAC inhibitors for 42hr,isolatedthe cell extracts,andthenassayed the luciferase activity. As shown in Figure 2A.

OCT4 gene expression is regulated by HDAC inhibitors. OCT4-Luciferase (OCT4-Luc) reporter plasmid was transfected intohuman keratinocytes (NHEKs). Six hours post-transfection, cells were treated with trichostatin A (TSA), sirtinol, or DMSOfor 42 hr. Relative luciferase activity per mg protein was on the Y-axis and the averages of fold of activity change were shown above each bar.

TSAslightlyenhanced luciferase activity by 1.2-fold (but not statistically significant), while sirtinolsignificantlyinhibitedOCT4-Luc expression by 12.5-fold. These data indicate thatsirtuins, but not class I/II HDACs, stimulate OCT4 expression.

To investigate a directinvolvement of sirtuins inOCT4 gene expression, we co-transfected SIRT1 expression plasmid pECE-SIRT1 with OCT4-Luc reporter plasmid into NHEK cellsfor two days. As shown in Figure 2B.

OCT4 gene expression is regulated by HDAC inhibitors. OCT4-Luciferase (OCT4-Luc) reporter plasmid was transfected intohuman keratinocytes (NHEKs). Six hours post-transfection, cells were treated with trichostatin A (TSA), sirtinol, or DMSOfor 42 hr. Relative luciferase activity per mg protein was on the Y-axis and the averages of fold of activity change were shown above each bar.

TSAslightlyenhanced luciferase activity by 1.2-fold (but not statistically significant), while sirtinolsignificantlyinhibitedOCT4-Luc expression by 12.5-fold. These data indicate thatsirtuins, but not class I/II HDACs, stimulate OCT4 expression.

To investigate a directinvolvement of sirtuins inOCT4 gene expression, we co-transfected SIRT1 expression plasmid pECE-SIRT1 with OCT4-Luc reporter plasmid into NHEK cellsfor two days. As shown in Figure 2B.

SIRT1 enhances OC4 expression.NHEK cells were co-transfected with OCT4-Luc reporter plasmid and the SIRT1 expression plasmid pECE-SIRT1 or pECE vector. Relative luciferase activity was assayed at2 days aftertransfection.SIRT1 deficiency abrogates OCT4 expression. Sirt1^+/+(wild-type) and sirt1^−/−(knockout) Mouse Embryonic Fibroblast (MEF) cells were transfected with the OCT4-Luc reporter plasmid for 2 days. Relative luciferase activity was indicated. Error bars indicate SD; n³ 3. Three stars indicate that the p-values are smaller than 0.001.

SIRT1 overexpression can enhance OCT4-Luc expression by 3.6-fold. To further confirm the role of SIRT1 on OCT4 expression, we compared OCT4-Luc expression in Sirt1^+/+ (wild-type) and sirt1^−/−(knockout) MEF cells. The expression of OCT4-Luc was nearly abrogated in sirt1^-/- MEF as compared to wildtype cells.  In the absence of SIRT1, luciferase activity was reduced by 18-fold. Our results indicate that SIRT1 is required for OCT4 gene transcription. 

SIRT1 Up-Regulates Plasmid-Derived and Endogenous OCT4 Pluripotency Regulators

Several integration-free methods have been used to generate iPSCs to avoid virus-integration induced mutagenesis. However, reprogramming efficiency using these methods is low in most cases. We elected to use the episomal plasmids developed by Okita, et al.[22] that contain pCXLE-hOCT3/4-shp53, pCXLE-hSK, and pCXLE-hUL (Figure1).Withtheseplasmids, iPSC generation is markedly enhanced by p53 suppression and the potent L-Myc[22]. We used human keratinocytes for iPSC generation because they are easy to obtain and their reprogramming efficiency is higher as compared to the human fibroblasts [44]. We also modified the reprogramming protocols shown in Figure 1. An extra transfection with Okitaplasmids with or without pECE-SIRT1was incorporated three days after the first transfection. After the second transfection, cells were maintained in NHEK medium for additional 11 days and then passaged intothetop of mitomycin-treated MEF feeders. The SIRT1 expression plasmid pECE-SIRT1 was co-transfected with the Okita plasmids into NHEK to examine the role of SIRT1 on reprogramming.

To confirm the expression of factors derived from the plasmids inthetransfected cells, we monitored their mRNA levels using sets of plasmid-specific PCR primers as listed in Table 1.mRNAsamples were isolated from transfected cells on days 3 and 7 after transfection.  In Okita plasmids + pECE-SIRT1 transfected NHEKs, SIRT1 mRNA increased ~300 and~900 fold on day 3 and day 7after transfection, respectively, as compared to cells transfected with Okitaplasmids+ pECEcloning vector(Figure 3,Tables 2 and 3).

NHEKs were co-transfected with Okita plasmids with or without SIRT1expression vector pECE-SIRT1 (or PECE cloning vector) as outlined in Figure 1. Controls are cells without any plasmidtransfection. RNA samples were isolatedon 3 or 7 days post transfection.  RNA (20 ng) was then subjected for RT-qPCR analyses with SIRT1and b-actinprimers as list in Table 1.The mRNA levels of SIRT1 were calculated relative to that of b-actinasDCt. The fold difference of mRNA levels of pECE-SIRT1 transfected cells (T) relative tonoplasmid control (C) cells was calculated according to the formula 2^{DCt(C)-DCt(T)}.  Error bars indicate SD; n = 3. 

The expression levels of OCT4, KLF4, SOX2, and L-MYC genes fromcells transfected with Okita plasmids alone varied widely (Figure 4 and Tables 2 and 3, compare Okita plasmids vs. controls).

NHEKs were transfected with Okita plasmids only (orange), Okita plasmids with pECE (green), or Okita plasmids with pECE-SIRT1(red) as outlined in Figure 1. Cells without plasmid transformation are used as controls (blue). RNA samples were isolated from cells on 3 or 7 days post transfection as indicated. RT-qPCR analyses were performed with respective primers (Table 1) to detect plasmid-derived mRNA (A and B) and endogenous mRNA (C and D).mRNA fold changes of indicated factors were calculated as described in Figure 3. Error bars indicate SD; n = 3.

On day 3 after transfection with Okita plasmids, the plasmid-encoded OCT4, KLF4, SOX2, and L-MYC were induced by 96, 261, 2678, and 499 folds, respectively, as compared to no plasmid controls (Table 2).

Thus, the induction of OCT4expression is the lowest and the induction of SOX2expression is the highest. The expression of OCT4 and L-MYC declined from day 3 to day 7 while the expression of KLF4 and SOX2 kept increasing within the same time interval. The reason for these differences is unclear given they are all under the same strong CAG promoter control [38](Figure 1).

When SIRT1 was co-transfected with Okita plasmids in NHEK, the mRNAs of plasmid-derived OCT4,KLF4, SOX2, and L-MYC were further increased by 3.8, 3, 3.9, and 5.9 folds on day 3 after transfection(Figure4 and Table 2, compare Okita plasmids + pECE-SIRT1 vs. Okitaplasmids + pECE).  On day 7 after plasmid transfection, the plasmid-derived OCT4, KLF4, SOX2, and L-MYCwere still induced by 5, 1.6, 2, and 2.9foldsin SIRT1 overexpressed cells(Figure 4 and Table 3). We also examined the endogenous gene expression of these four pluripotency regulators. Interestingly, the endogenous OCT4 was induced by5.6 and 3.4 folds on days 3 and 7, respectively, while the other three endogenous gene expressions were not enhanced by SIRT1 overexpression(Figure 4, and Tables 2 and 3). These results indicate that SIRT1 can stimulate transcription of endogenous OCT4 gene and genes under the CAG promoter control.

SIRT1 Enhances iPSC Generation

NHEK cells transfected with the Okita plasmids were grown on top of mitomycin-treated MEF feeders for more than 30 days to generate iPSCs.  At days 1 and 15, NHEK cells did not exhibit any change in morphology before and after transfection (Figure 5).

NHEKs were transfected with Okita plasmids only, Okita plasmids +pECE, or Okita plasmids + pECE-SIRT1 as outlined in Figure 1.Imagrs of one iPSC-like colony from Okita plasmids only and two iPSC-like colonies from Okita plasmids + pECE-SIRT1. Images of NHEKs at days 0 and 15 and iPSCcoloniesdays 33-40 after transfectionwereshown. (B) Numbers of iPSC-like colonies obtained per 10⁵NHEK cells seeded. Colony number was scored on day 30 after plasmid transfection. No colonies were observed from cells withoutOkita plasmid DNA transfection. Error bars indicate SD; n = 3.  One and two stars indicate that the p-values are smaller than 0.05 and 0.01, respectively.

iPSC-like colonies(Figure 5) were observed in Okita plasmid transfected cells but not in controls without plasmids. ThenumberofiPSC-like coloniesincreased by7-fold fromNHEKcells co-transfected with pECE-SIRT1 plasmid plus Okita plasmids as compared with that from cells co-transfected with cloning vector plus Okita plasmids (Figure 5).

The iPSC-like colonies derived from pECE-SIRT1 plusOkita plasmids last longer than those derived from Okita plasmids alone (Figure 5).To confirm that these colonies are bona fide iPSCs, we checked whether they express the key stem cell markers TRA-1-60 [39] and OCT4 [10,11].  The colonies were stained with Alexa Fluor 594 anti-human TRA-1-60-Rantibody and OCT4 antibody followed by Alexa Fluor 488goat anti-mouse IgGantibody.As observed under a fluorescence microscope, iPSC-like colonies express TRA-1-60-R (green fluorescence)and OCT4 (green fluorescence), indicating they are iPSCs. Both MEF feeder cells and iPSC-like colonies are stained with DAPI, however,MEF feeder cells do not express TRA-1-60-R and OCT4.

Discussion

The therapeutic potential of iPSCs has been hampered by low reprogram efficiency, potential mutagenesis associated with viral-mediated reprogramming, and high genome instability. It has been shown that reprogramming can be facilitated by inhibitors of classes I/II HDACs and DNMTs[13-17].  SIRT1 is a potential reprogramming factor because it regulates acetylation levels of histones and many transcription factors (reviewed in [23]. In this report, we have shown that expression of SIRT1 can enhance endogenous OCT4 gene expression in human keratinocytes. In addition, SIRT1 overproduction enhances the transcription of all plasmid-derived reprogramming factors under the CAG promoter control. Moreover, iPSC-like colonies are generated more efficiently when SIRT1 is overexpressed. Our result of SIRT1 enhancement on human iPSC generation is consistent with previous reports that mouse Sirt1 can facilitate iPSC generation from mouse embryonic fibroblasts [32,35]. Our data indicate that SIRT1 enhances somatic cell reprogramming, in part, through enhancement of gene expression of endogenous OCT4 and ectopic transcription factors.

However, the molecular mechanism by whichSIRT1 enhances the OCT4 and CAG promoter activities is unclear. As a histone deacetylase, SIRT1 would act to silence gene transcription (reviewed in [23]. This is certainly not in line with our findings that SIRT1 activates endogenous OCT4 and CAG-regulated genes.It is possible that SIRT1 activates gene expression through deacetylatingor stimulating transcription factors, p53, and DNA repair enzymes [45].One likely scenario is that SIRT1 enhances the demethylation activity of Thymine DNA Glycosylase(TDG) and thus activates genetranscriptionbecause TDG can be deacetylatedandstimulated by SIRT1 [46]. 

Since SIRT1 stimulates the expression ofOCT4and NANOGandexerts substantial enhancement on reprogramming(this report and [32,35,47], future development using SIRT1 activators and small molecules [10,11]may eliminate the requirement of any transcription factors for iPSC production. One potential SIRT1 activator is resveratrol (a natural phenol) [48].  Indeed, resveratrol has been shown to promote iPSC formation [49,50].However, its direct activation of SIRT1 has been refuted several times [51-53].Ghosh et al.[54]have provided a mechanistic explanation to this controversy. These authors have shown that resveratrol activates SIRT1 by increasing its binding with lamin A, thus facilitating SIRT1 localization to nuclear matrix. They have also shown that the rescue of adult stem cell decline by resveratrol is SIRT1-dependent.  Resveratrol may be combined with Valproic acid (VPA, a class I/II HDAC inhibitor) and/or 5-azacytidine (azaC, a DNMT inhibitor) [13-17]to achieve optimal reprogramming.

Our novel approaches will not only increase the efficiency of iPSC induction but also generate iPSCs with high genomic stability. The genomic stability ofiPSCs should be stringently maintained for the safety in tissue replacement applications[8]. SIRT1 plays a pivotal role in controlling genomic stability by deacetylating DNA repair enzymes and p53 [24-28,45]. By overproducing SIRT1 or using SIRT1 activators, the derived iPSCs should be highly proliferative, tolerant to oxidative stress and DNA damage, and highly similar to embryonic stem cells. Our finding will take steps closer to generate high quality iPSCs for regenerative medicine.

Acknowledgment

This work was supported by Maryland Stem Cell Research Fund (2014-MSCRF-0656).

Figure 1: Protocol for iPSC generation.

Figure 2: Effects of HDAC inhibitors and SIRT1 on OCT4 gene expression.

Figure 3: SIRT1 mRNA expression after pECE-SIRT1 plasmid transfection during iPSC generation.

Figure 4: Gene expression of OCT4, KLF4, Sox2, and L-MYC in Okita-plasmid transfected NHEKs.

Figure 5: SIRT1 Enhances iPSC Generation.

Figure 6: iPSC-like colonies express the key pluripotent stem cell markers.

                    Table 1:Oligonucleotides used for qPCR.

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