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1 Center for iPS Cell Research and Application (CiRA), Institute for Integrated Cell-Material Sciences, Kyoto University, Kyoto 606-8507, Japan
2 Department of Stem Cell Biology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto 606-8507, Japan
3 CREST and Yamanaka iPS Cell Special Project, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan
4 Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA
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Abstract |
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Introduction |
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We analyzed mouse expressed sequence tag databases and identified genes specifically expressed in undifferentiated ES cells, which we designated ES cell associated transcripts (ECATs) (Takahashi et al. 2003; Tokuzawa et al. 2003; Maruyama et al. 2005; Amano et al. 2006; Imamura et al. 2006). One of the ECATs was Nanog, which is now considered to be one of the key transcription factors in maintaining pluripotency of ES cells (Chambers et al. 2003; Mitsui et al. 2003). Together with Oct3/4, Sox2 and several other proteins, Nanog constitutes a transcription factor network that plays central roles underlying the undifferentiated state in ES cells and early embryos (Boyer et al. 2005; Masui et al. 2007; Niwa 2007).
Another ECAT encodes Sall (Sal-like) 4, which belongs to the Spalt (Sal) transcription factor family characterized by highly conservative C2H2 zinc-finger motifs (Kohlhase et al. 2002a). Spalt was initially identified in Drosophila as a homeotic gene required for the early development of the anterior head and posterior tail areas (Jurgens 1988; Kuhnlein et al. 1994). Homologues of Spalt were later found in Caenerhabditis elegans, Xenopus, fish, chick, mouse and human (Sweetman & Munsterberg 2006). The mouse and human have four Sall proteins (Sall1–4) (Sweetman & Munsterberg 2006). In human, mutations at the SALL4 locus cause an autosomal dominant disorder known as Okihiro syndrome, which is characterized by limb deformity, eye movement deficits and, less commonly, anorectal, ear, heart, cranial midline and kidney anomalies (Kohlhase et al. 2002b, 2005; Miertus et al. 2006). Similar phenotypes were reported in Sall4 heterozygous mutant mice (Koshiba-Takeuchi et al. 2006; Sakaki-Yumoto et al. 2006; Warren et al. 2007).
Knockdown of Sall4 results in the loss of the undifferentiated state in ES cells and differentiation into trophectoderm-like cells, suggesting that Sall4 contributes to self-renewal of ES cells (Wu et al. 2006; Zhang et al. 2006). In ES cells, many binding sites of Sall4 overlapped with those of Oct3/4, Sox2 and Nanog, as determined by ChIP on chip analyses (Lim et al. 2008; Yang et al. 2008). Sall4-null mouse embryos die shortly after implantation (Elling et al. 2006; Sakaki-Yumoto et al. 2006). Until now, the generation of ES cells from Sall4-null blastocysts has not been reported. Therefore, the role of Sall4 in ES cell generation from the ICM remains to be determined.
In 2006, ES cell-like pluripotent stem cells, which were designated induced pluripotent stem (iPS) cells, were directly generated from mouse fibroblasts by the introduction of four transcription factors (Oct3/4, Sox2, Klf4 and c-Myc) (Takahashi & Yamanaka 2006; Okita et al. 2007). The same factor combination or a partially different combination (OCT3/4, SOX2, NANOG and LIN28) can generate iPS cells from human fibroblasts (Takahashi et al. 2007; Yu et al. 2007). Direct reprogramming can be achieved even without Myc transgenes, albeit with a lower efficiency, both in mouse and human using the remaining three factors, Oct3/4, Sox2 and Klf4 (Nakagawa et al. 2008; Wernig et al. 2008). The role of Sall4 in iPS cell generation has not yet been elucidated.
This study investigated the role of Sall4 during the establishment of pluripotent stem cells from blastocysts (ES cells) and from somatic cells (iPS cells). Sall4 has positive roles in the generation of both types of pluripotent stem cells. However, the role of Sall4 can be compensated, at least in part, by Sall1 and other factor(s) yet to be determined.
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Results |
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RT-PCR analyses were employed to determine the expression of Sall4 and Sall1 genes in mouse ES cells and adult tissues. Two lines of mouse ES cells, RF8 and MG1.19 highly expressed both Sall1 and Sall4 in the undifferentiated state (Fig. 1a). The expression levels decreased upon differentiation induced by retinoic acid. Sall4 is also expressed in the testis and ovary, albeit at lower levels than in undifferentiated ES cells. Trace amounts of Sall4 mRNA were detected in the brain, heart and muscle of adult mice. In contrast, the expression of Sall1 was detected in kidney, at a comparable level to that in undifferentiated ES cells.
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Neural tube defect, anorectal malformations and peri-implantation lethality in Sall4 mutant mice
The disruption of the exon 2 by the IRES-β-geo cassette results in deletion of ~95% of the C-terminus of the Sall4 protein. Heterozygous intercrosses did not yield any Sall4-null mice (Table 1). In addition, the numbers of Sall4 heterozygous mutant mice were fewer than expected according to the Mendelian law. The partial lethality of Sall4 heterozygous mice was confirmed from crosses of heterozygous mice and wild-type animals 4–5 weeks after birth (Table 1). Out of 129 surviving heterozygous mice, 12 showed anorectal malformation and died within a few months after birth (Supporting Fig. S2a–b). Approximately equal numbers of wild-type embryos and heterozygous mutants were found at E11.5–14.5. However, ~16% of Sall4 heterozygous embryos showed a failure of neural tube closure at the forebrain/midbrain boundary (Table 1; Supporting Fig. S2c). The numbers of Sall4 heterozygous embryos at E18.5 were slightly fewer than those of wild-type embryos, with three out of 19 heterozygous mutants showing exencephaly. These phenotypes of Sall4 haploinsufficiency were in accordance with previous reports (Sakaki-Yumoto et al. 2006; Warren et al. 2007). In addition, we found that ~50% of Sall4 heterozygous embryos at the 15- to 20-somite stages did not undergo closure of the forebrain/midbrain and hindbrain region where Sall4 is expressed, while 95% of Sall4 wild-type embryos completed the closure (Fig. 1e).
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Role of Sall4 in ES cell generation from Sall4-null embryos
To understand the roles of Sall4 in peri-implantation embryos and in ES cell generation, E3.5 blastocysts from heterozygous intercrosses were cultured in vitro. Within seven days in a feeder-free culture, Sall4 wild-type and Sall4 heterozygous blastocysts hatched from the zona pellucida and then developed trophectodermal outgrowths and proliferating ICM. However, Sall4-null blastocysts showed delayed hatching and slower proliferation of ICM and primitive endoderm (Fig. 2a, Supporting Fig. S3b). A TUNEL (TdT-mediated dUTP-biotin Nick End Labeling) assay showed that apoptosis did not increase in Sall4-deficient ICM compared to wild-types or heterozygotes (Supporting Fig. S3c).
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The two Sall4-null ES-like cells were similar, but not identical to wild-type ES cells. They expressed normal amounts of Oct3/4, and other pluripotency genes (Fig. 3a,b). The expression levels of Sall1 were slightly higher in the two Sall4-null cells than in the heterozygous clones (Fig. 3a,b). The global gene expression patterns of the two Sall4-null clones varied slightly more than did those of the two heterozygous clones, as revealed by DNA microarray analyses (Supporting Fig. S4c). Their morphology was similar to that of ES cells, but both clones showed a tendency to spontaneously differentiate when cultured on gelatin-coated dishes in the presence of LIF (Fig. 3c). This tendency was confirmed by a colony assay, which showed that Sall4-null clones resulted in higher ratios of differentiated colonies when plated at a clonal density than did wild-type ES cells (Fig. 3d). Both the Sall4-null clones displayed reduced proliferation in comparison to the wild-type ES cells (Fig. 3e). In addition, teratoma formation was impaired (Fig. 3f). Teratoma formation was partially restored by the introduction of the Sall4 cDNA in clone #1.
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Role of Sall4 in mouse iPS cells generation
To determine whether Sall4 plays a role during mouse iPS cell generation, short hairpin (sh) RNA against the mouse Sall4 gene was introduced along with the three reprogramming factors (3F: Oct3/4, Sox2 and Klf4) into mouse embryonic fibroblasts (MEFs), thus carrying the EGFP-IRES-puro cassette driven by Nanog promoter. The two shRNAs, designed from different target sites of Sall4, induced a partial reduction of Sall4 proteins (Supporting Fig. S5). The number of EGFP-positive colonies significantly decreased by the Sall4 shRNAs at 28 days post-transduction (Fig. 4a). The effect of Sall4 shRNA (#1) was rescued by the forced-expression of human SALL4 (hSALL4) cDNA, which did not have the target sequences of mouse Sall4 shRNAs. The hSALL4 cDNA also tended to rescue the effect of Sall4 shRNA (#2), but the change was not statistically significant.
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Effects of ectopic expression of Sall4 in human iPS cell generation
Next, the role of SALL4 in human iPS cell generation was investigated. The SALL4 retrovirus together with the three reprogramming factors was introduced into human dermal fibroblasts (HDFs) derived from persons of various ages. Flat and tightly packed colonies were observed approximately 25 days after transduction. These colonies resembled human ES (hES) cells. The number of iPS cell colonies was counted 40 days post-transduction. The numbers of hES-like colonies from HDFs derived from a 36-year-old female were increased by the addition of SALL4 approximately tenfold in comparison to 3F + Mock (Fig. 5). In contrast to the c-MYC retrovirus which increases both hES cell-like colonies and non-iPS cells colonies when added in addition of 3F, SALL4 only increased hES cell-like colonies (data not shown). These cells continued to show morphology similar to that of hES cells with subsequent passages (Supporting Fig. S6a,b). RT-PCR showed that the cells expressed hES-cell marker genes (Supporting Fig. S6c). They were able to differentiate into three germ layers that were positive for 
-smooth muscle actin (SMA), βIII-tubulin and -fetoprotein (AFP) by embryoid body formation (Supporting Fig. S6d–f). These data confirmed that these hES cell-like cells were iPS cells.
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To investigate the possible causes of the variable effects of SALL4 on different HDF lines, the expression levels of endogenous SALL4 were determined in each fibroblast line. DsRed was introduced as a control, or the three reprogramming factors into the five different HDF lines used in Fig. 5. The fibroblast line from a 36-year-old female, which showed the strongest effect of the SALL4 transgene, had the lowest expression level of the endogenous SALL4 (Supporting Fig. S7).
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Discussion |
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Previous reports have shown that ICM derived from Sall4-deficient embryos fail to proliferate (Elling et al. 2006; Sakaki-Yumoto et al. 2006), and no ES cell lines were established from Sall4-deficient blastocysts (Elling et al. 2006). We also showed the impaired proliferation of Sall4-null ICM. However, two lines of Sall4-null ES-like cells were established from blastocysts, albeit with lower efficiency and a much slower time course. This result showed that Sall4 plays an important role in the generation of mouse ES cells, but its functions can occasionally be compensated by other factors.
The Sall4-null ES-like cells are similar, but not identical to wild-type ES cells. They are morphologically similar to wild-type ES cells. They express many ES cell marker genes at comparable levels to those in ES cells. However, Sall4-null cells showed a slower proliferation and a tendency to spontaneously differentiate. When plated at a clonal density, the number of alkaline phosphatase positive, Sall4-null colonies was less than one fourth of that of wild-type. Sall4-null ES-like cells could form only very small or no teratomas.
A possible candidate of the factors that restore the depletion of Sall4 is Sall1. The amino acid sequences in the two Sall proteins are 36% identical. Furthermore, the C2H2 zinc finger domains are 78–89% identical in the two proteins. Both Sall1 and Sall4 are highly expressed in undifferentiated ES cells. Sall1 and Sall4 have synergistic functions during neural, heart, renal and anorectal development (Sakaki-Yumoto et al. 2006; Bohm et al. 2008). Furthermore, Sall1 seems to be involved in regulating the expression of Nanog in place of Sall4 in Sall4-null ES-like cells. However, a reduction of Sall1 did not led to any change in the expression of Oct3/4 or Sox2 in Sall4-null ES-like cells. Therefore, it is likely that Sall1 can compensate for only a part of the Sall4 functions.
Prior to our report, two groups have reported the effect of Sall4 in mouse ES cells (Sakaki-Yumoto et al. 2006; Zhang et al. 2006). All the three groups found that Sall4-suppressed ES (-like) cells showed impaired proliferation. However, we and Sakaki-Yumoto et al. did not observe decrease in Oct3/4 or increase in trophectoderm marker genes such as Cdx2, which were reported by Zhang et al. (2006) (Lim et al. 2008). One possibility of the different phenotype is that we and Sakaki-Yumoto et al. used gene targeting, whereas Zhang et al. used shRNA to suppress Sall4. The shRNA-mediated suppression is much quicker than that by gene targeting, thus compensation mechanisms could not occur.
Mouse Sall4 has a positive role not only in ES cell generation, but also in iPS cell generation. The suppression of endogenous Sall4 expression by shRNA decreased the efficiency inducing of iPS cell from mouse somatic cells and the efficiency was restored by re-introducing hSALL4. These results are consistent with the report that ectopic expression of Sall4 enhanced the reprogramming of MEF by cellular fusion with ES cells (Wong et al. 2008).
SALL4 showed variable effects on human iPS cell generation. In some HDFs, SALL4 consistently showed a positive effect, as in mouse ES cells and iPS cells. In other HDFs lines, however, SALL4 did not show such an effect. The mechanisms of this variable effect remain to be determined. One possible mechanism is different basal expression levels of the endogenous SALL4 gene in each HDF line. Endogenous SALL4 was expressed at the lowest level in human fibroblast lines with the strongest effect of exogenous SALL4. The finding that each HDF line can differ substantially has to be considered when conducting studies to explore the effects of genes or other factors on iPS cell generation.
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Experimental procedures |
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PCR was carried out with ExTaq polymerase (Takara, Japan) for Sall4 and Nat1 and KOD-plus polymerase (TOYOBO) for Sall1. Sall1 was amplified with primers S (5'-CAC CAT GTC ACG GAG GAA GCA AGC GAA GC-3') and AS (5'-TTA CAA GGG GTT GGC AGA TGT TCG TAA A-3'). Sall4 was amplified with primers S (5'-CCG AGA CCC TGA AAT TGC AGC AAC TA-3') and AS (5'-ACG AGA AGT TCT TTC CAC ACC GTG TG-3'). The primer sequences of Nat1 are shown in Supporting Table S1, which is available in the online data supplement.
Generation of Sall4-deficient mice
To make the 5' and 3' homologous region of the targeting vector, a fragment of the 5' flanking region containing exon 1 and exon 2 and a 2-kb fragment of the 3' flanking region containing exon 3 were amplified from genomic DNA of RF8 ES cells derived from 129SV/Jae. The 6.5-kb 5' arm was amplified with sense primer (5'-GCG GCC GCC GTC TTA CCC TTT CAA AAC CTT-3') and antisense primer (5'-CCG GGG CCG CTT CAC TAG TAT GGA GTC CTC-3'). A 2-kb 3' arm was amplified with sense primer (5'-GGA TCC TTT CGT GTG TAA CAT ATG GGG-3') and antisense primer (5'-CTC GAG AGA GGG TTT CAT CAG ATG GTC C-3'). These PCR products were cloned into the pCR2.1 vector (Invitrogen). The 5' and 3' arms were cloned into the NotI/SpeI sites and the BamHI/XhoI sites of the pBSSK(-)IRES-βgeo-pA vector. The targeting vector was linearized with NotI and introduced into RF8 ES cells by electroporation. Among the 72 G418-resistant colonies screened, Southern blotting showed six to be correctly targeted. Two clones were injected into C57 BL/6J blastocysts, thus yielding chimeric males that transmitted the targeted allele through the germ line from one clone. Chimeras were crossed with C57 BL/6J mice to generate Sall4 heterozygous mice. Clones with homologous recombination were screened by a Southern blot analysis as previously described. The 5' probe was amplified with a sense primer (5'-CGA AGG AGG CTA AAA TTT CCC AAC TC-3') and an antisense primer (5'-AAG TTA CCG GCG TAG GTG GGT GCT TA-3'). Hybridization with this probe with NsiI-digested genomic DNA yielded a 12-kbp band from the wild-type locus and a 20-kbp band from the targeted locus. A 3' probe, amplified with sense primer (5'-CAT GGC CAA ACA CCA GTT CCC TCA CTT C-3') and antisense primer (5'-AAG ACA GGG TCT CGA GGC ATA AAA CCA G-3'), detected a 6.4-kb band from the wild-type locus and a 20-kb band from the targeted locus. Once the homologous recombination was confirmed by a Southern blot analysis, cells, the embryos and mice were genotyped using PCR with a sense primer (5'-CAA TCC TCC TGA GTC AGG ACA TGT GC-3'), and antisense primers (5'-CTG CAG AGT CAC ATT GGT GTT GGC TA-3') and (5'-GCG GAA TTC TCT AGA GTC CAG ATC-3'). PCR with these primers produced a 510-bp fragment from the wild-type locus and a 180-bp fragment from the targeted locus. X-gal staining of embryos was performed as described earlier (Avilion et al. 2003).
Plasmid construction
The open reading frame (ORF) of mouse Sall4 was amplified by RT-PCR and subcloned into pENTR-D-TOPO (Invitrogen). To obtain the ORF of hSALL4, Homo sapiens cDNA FLJ10804 (AK001666 [GenBank] ) was provided by NBRC, NITE-DOB, Kisarazu, Japan. The deletions of 2 bp of AK001666 [GenBank] clone were corrected by PCR-based site-directed mutagenesis, resulting in coding of full-length hSALL4 protein. After sequence verification, ORF of hSALL4 was amplified from the plasmid and then cloned into pENTR-D-TOPO (Invitrogen). All of the genes subcloned into pENTR-D-TOPO were transferred to pMXs and pCAG-IRES-puro (pCAG-IP) using the Gateway cloning system (Invitrogen), according to the manufacturer's instructions. For pMXs-U6-Sall4 shRNAs, annealed oligos were subcloned into the BfuAI site of pU6-puro. The fragments of U6-shRNA were removed by BamHI digestion from pU6-shRNA-puro and subcloned into the BamHI site of pMXs. All of the fragments were verified by sequencing. The primer and oligo sequences are shown in Supporting Table S1.
Blastocyst culture and establishment of ES cells from blastocysts
Sall4 heterozygous mice were intercrossed and blastocysts were flushed from the uteri at E3.5. The blastocysts were cultured individually on gelatin-coated four-well plates in ES medium containing LIF under the stereomicroscope and photographed everyday for 7 days. For PCR-genotyping, blastocyst outgrowth was lysed in 10 µL of lysis buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.0, 2.5 mM MgCl2, 0.45% NP40, 0.45% Tween20, 0.2 mg/mL proteinase K) at 56 °C for 6 h. For the establishment of ES cells, blastocysts were collected from Sall4 heterozygous intercrosses at E3.5 and individually cultured on four-well plates with mitomycin C-treated SNL feeder cells in TX-WES medium (Cosmo Bio, Japan) (Schoonjans et al. 2003). After 5–8 days, the undifferentiated cell mass were transferred to new four-well plates on SNL feeder cells. After an additional 7–20 days, undifferentiated colonies were transferred individually to new plates with SNL feeder cells. After 2–15 days, undifferentiated ES cells were passaged and maintained in ES medium containing LIF (Meiner et al. 1996).
Colony formation, proliferation and rescue analysis of ES cells
For the colony formation assay, the cells were plated at 500 cells per well of gelatinized six-well plates in the presence of LIF. After 5 days, the cells were stained with the Alkaline Phosphatase Kit (Sigma-Aldrich). For the proliferation assay, 1 x 104 cells were plated per well of a 24-well plate with SNL feeder cells. After 6 days, the cells were counted using a Z2 Coulter Counter (Beckman Coulter). For the rescue analysis of Sall4-null ES-like cells, either the linearized pCAG-IP vector (negative control) or the Sall4 expression vector (Sall4 in pCAG-IP) was introduced into Sall4-null ES-like cells by electroporation. The cells were selected with 2.0 µg/mL puromycin on puromycin-resistant SNL cells for 10 days. Several clones were established on gelatin-coated dishes, and then were used for the experiments.
Microarray analysis
Total RNA from Sall4 heterozygous and Sall4-null ES-like cells, two lines, respectively, and Fbx15-null MEFs, as using a negative control of pluripotent markers (Takahashi & Yamanaka 2006), was labeled with Cy3. Samples were hybridized with Mouse Oligo Microarray (G4121B, Agilent). Each sample was hybridized once with the one color protocol. The arrays were scanned with a G2565BA Microarray Scanner System (Agilent). The data were analyzed using GeneSpring the GX10.0.1 software program (Agilent). The normalization procedures were applied; each chip was normalized to the 50th percentile of the measurements taken from that chip. The flag settings were set as described below. Feature: not uniform (M), a population outlier (M), saturated (M), not positive and significant (A), not above background (A). In the genes with a "Present or Marginal" flag value, at least one out of the five samples was used for the analyses (15 530 genes).
Knockdown analysis with siRNA
Stealth small interference RNA (siRNA) against mouse Sall4 [three RNAi; Sall4siRNA#1 MSS246806_contorl duplexes (N.C.) Med, #2 MSS246807_N.C. Med and #3 MSS246808_N.C. Low], Sall1 (three RNAi; Sall1siRNA#1 MSS226691_N.C. Low, #2 MSS226692_N.C. Med, #3 MSS226693_N.C. Med) and RNAi negative control duplex (two RNAi; Med GC and Lo GC) were purchased from Invitrogen (Carlsbad, CA). The control duplexes were used according to the recommendations by Invitrogen in June 2006. A population of 4.0 x 105 cells was transfected with the siRNA (final concentration 40 nM) against Sall4, Sall1 or negative control RNA (N.C. Med and Low) using Lipofectamine RNAiMAX according to a reverse transfection protocol per well on the gelatin-coated six-well plate. At 48 h after transfection, the total RNA was isolated with Trizol (Invitrogen) and then it was treated with the Turbo DNA-free kit (Ambion) to remove any genomic DNA contamination. One microgram of total RNA was used for the reverse transcription reaction with ReverTraAce- (Toyobo, Japan) and dT20 primer, according to the manufacturer's instructions. qPCR was performed with Platinum SYBR Green qPCR Supermix UDG (Invitrogen) and then it was analyzed with the 7300 real-time PCR system (Applied Biosystems). The mRNA levels were normalized against the average for β-actin and Nat1 mRNA. The primers sequences are shown in Supporting Table S1.
Western blotting
Rabbit polyclonal antiserum against mouse Sall4 was generated against the first 300 amino acids of mouse Sall4. Half a million cells were lysed with 100 µL of 1 x SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue, 5% β-mercaptoethanol). The cell lysates were vortexed and heated for 10 min at 100 °C. The cell lysates (10 µL) were separated by electrophoresis. Western blotting was performed as described earlier (Takahashi et al. 2007). The antibodies used included anti-Sall4 (1 : 1000), anti-SALL4 (1 : 500, abcam), anti-Sall1 (1 : 1000, PPMX), anti-Oct3/4 (C10, 1 : 500, Santa Cruz) and β-actin (1 : 4000, Sigma).
Generation of iPS cells from somatic cells
The retroviral transduction and iPS cell generation were performed as previously described with some modifications (Takahashi et al. 2007; Nakagawa et al. 2008). Briefly, PLAT-E cells were plated at 3.6 x 106 cells per 100 mm dish and incubated overnight. The cells were transfected with pMXs-based retroviral vectors by using Fugene 6 transfection reagent (Roche). Two days after transfection, virus-containing supernatant was collected and filtered with a 0.45 µm cellulose acetate filter. MEFs that contained both the Nanog-EGFP-IRES-Puror reporter and the Fbxo15-βgeo reporter were used for iPS cell generation (Okita et al. 2007). For the Sall4 knockdown analysis during the iPS cell generation from MEFs, retrovirus-containing supernatants for Oct3/4, Sox2, Klf4, Sall4 shRNA, and Mock (as a control) or hSALL4, were mixed with equal amounts of each virus. For an analysis of the effects of Sall4 transgene during the generation of iPS cells from MEFs, retrovirus-containing supernatants of Oct3/4, Sox2, Klf4, along with Mock or Sall4, were mixed with equal amounts of each virus. For transfection, MEFs were seeded at 1 x 105 cells per well of a six-well plate. MEFs were incubated in the virus/polybrene-containing supernatants for 24 h. Four days after transduction, MEFs transduced with the factors were reseeded at 2 x 105 cells per well of a six-well plate with SNL feeder cells and cultured in ES medium. The selection with puromycin (1.5 µg/mL) was started at 21 days after transduction. Twenty-eight days after transduction, the colonies were counted. Some colonies were then isolated for expansion. For analysis of the effects of the SALL4 transgene during the generation of iPS cells from HDFs, retrovirus-containing supernatants of OCT3/4, SOX2, KLF4, along with Mock or SALL4, were mixed with equal amounts of each virus. The iPS cell generation from HDFs was performed as previously described (Takahashi et al. 2007). HDFs from several lines were purchased from Cell Applications, Inc. and the Japanese Collection of Research Bioresources (JCRB). The detail information on the HDF lines is shown in Supporting Table S2, which is available in the online data supplement. Forty days after transduction, the number of human ES-like colonies was counted. Some colonies were then picked up and used for further analyses.
Characterization of iPS cells and HDFs
RT-PCR and in vitro differentiation of human iPS cells were performed as described earlier (Takahashi et al. 2007). For the expression levels of endogenous SALL4 during the generation of iPS cells, retrovirus-containing supernatants for DsRed alone or the factors consisted of OCT3/4, SOX2, KLF4 and Mock were used for the transduction. Total RNA was isolated 6 days after transfection. The isolation of total RNA, DNase reactions and reverse transcription reactions was performed as mentioned before. qPCR was performed with SYBR Premix Ex TaqII (Takara) and analyzed with the 7300 real-time PCR system (Applied Biosystems).The data were normalized with those of G3PDH. The primers are shown in Supporting Table S1.
Statistical analyses
The data are presented as the averages and standard deviations of independent biological replicate experiments. One-way ANOVA (Fig. 2c) or one-way repeated measures ANOVA (figures excluding Fig. 2c), followed by Bonferroni post hoc test, was carried out using KaleidaGraph.
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Acknowledgements |
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Footnotes |
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* Correspondence: yamanaka{at}frontier.kyoto-u.ac.jp
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References |
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Accepted: 2 March 2009
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