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Suppression of ES cell differentiation by retinol (vitamin A) via the overexpression of Nanog
r 2007, Copyright the Authors Differentiation (2007) 75:682–693 DOI: 10.1111/j.1432-0436.2007.00169.x Journal compilation r 2007, International Society of Differentiation
OR IGI N A L A R T IC L E
Liguo Chen . Minying Yang . Joyce Dawes . Jaspal Singh Khillan
Suppression of ES cell differentiation by retinol (vitamin A) via the overexpression of Nanog
Received November 13, 2006; accepted in revised form January 4, 2007
Abstract Embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts maintain their pluripotency through a complex interplay of different signaling pathways and transcription factors including Leukemia Inhibitory Factor (LIF), homeo-domain protein Nanog and POU-domain-containing transcription factor Oct3/4. LIF can maintain the self-renewal of mouse ESCs by activating the Jak/Stat3 pathway; however, it is dispensable for human ESCs. Nanog, a homeo-domain transcription factor alone is sufficient for sustaining the self-renewal of ESCs. Overexpression of Nanog by heterologous promoters can maintain selfrenewal of human and mouse ESCs in the absence of LIF/Stat3 pathway. The mechanisms that control the expression of Nanog, however, remain poorly understood. In this report we demonstrate that retinol, the alcohol form of Vitamin A, can suppress the differentiation of ESCs by up-regulating the expression of Nanog. Retinol is mainly associated with differentiation through its active metabolite retinoic acid during early development of the embryo. The activation of Nanog by retinol is not mediated via retinoic acid signaling and appears to be independent of previously described LIF/ Stat3, bone morphogenic proteins, Wnt/b-catenin, and Oct3/4-Sox2 pathways. These studies therefore, reveal a previously unknown function of retinol and offer a model system to define alternate regulatory pathways that control the self-renewal of ESCs as well as to identify upstream ‘‘master’’ regulatory factors that are responsible for maintaining the integrity of stem cells.
Liguo Chen Minying Yang Joyce Dawes Jaspal Singh . ) Khillan (* Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. Tel: 11 412 383 6987 Fax: 11 412 865 8695 E-mail: [email protected]
Key words self renewal of ES cells overexpression of Nanog retinol (Vitamin A) retinoic acid suppression of ES cell differentiation Oct3/4
Introduction Embryonic stem cells (ESCs) derived from the inner cell mass (ICM) of mammalian blastocysts have the potential to grow indefinitely while maintaining pluripotency. After injection into blastocysts, ESCs can contribute to all the primary germ layers such as endoderm, mesoderm, and ectoderm (Evans and Kaufman, 1981; Martin, 1981). Depending upon the culture conditions, these cells can differentiate into different cell types (Smith, 2001). Because of these properties, ESCs will hopefully be useful in treating degenerative diseases such as Parkinson’s disease, spinal cord injury, diabetes, and cardiovascular diseases in humans (Thomson et al., 1998). ESCs maintain pluripotency by expressing a specific transcriptional program that suppresses their differentiation. Leukemia inhibitory factor (LIF), a member of IL6 family of lymphokines, can maintain the self-renewal of ESCs by activating Stat3 (Smith et al., 1988; Williams et al., 1988; Stewart et al., 1992; Smith and Crompton, 1998). LIF binds to a heterodimer receptor, consisting of LIFR and GP130 that lead to the activation of Jak/Stat3 pathway (Ernst et al., 1996; Ihle et al., 1996). This pathway then activates the downstream genes (Davis et al., 1994). Although LIF signaling is important for mouse ESCs (Burdon et al., 2002), it is not sufficient to sustain their self-renewal and requires additional signals. These signals are provided by the bone morphogenic proteins (BMPs), which activate inhibitors of differentiation (Id) genes (Ying et al., 2003). LIF/Stat3 signaling, however, is dispensable for human ESCs (Daheron et al., 2004) suggesting that this pathway is not fundamental for maintenance of pluripoten-
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cy. Furthermore, Wnt/b-catenin signaling has also been implicated in maintaining the undifferentiated status of both human and mouse ESCs (Saito et al., 2004). POU domain-containing transcriptional factor Oct3/ 4, homeo-box genes nanog and sox2 and fibroblast growth factor 4 (fgf4) play a key role in maintaining the pluripotency of ESCs (Yuan et al., 1995; Wilder et al., 1997; Nichols et al., 1998; Avilion et al., 2003; Chambers et al., 2003; Mitsui et al., 2003). The oct3/4 and nanog are specifically expressed in ICM and epiblast of the early embryo (Nichols et al., 1998; Avilion et al., 2003; Chambers et al., 2003; Mitsui et al., 2003). Null mutations of oct3/4 (Nichols et al., 1998) and nanog (Mitsui et al., 2003) result in early embryonic lethality. Nanog alone, however, can maintain the self-renewal and pluripotency of ESCs independent of LIF/Stat3 pathway (Chambers et al., 2003; Mitsui et al., 2003). Down-regulation of Nanog results in the differentiation of ESCs into extra embryonic endoderm, whereas overexpression of Nanog allows propagation and self-renewal of ESCs in the absence of LIF (Darr et al., 2006). Although many signaling pathways are involved in the self-renewal of ESCs (Smith, 2001), only limited information is available about the regulatory mechanisms that control the key genes responsible. An understanding of the circuitary of transcription factors that maintains the pluripotency of ESCs is fundamental to realizing their full potential for therapeutic applications. The overexpression of Nanog by heterologous promoters has been used as a model to study the selfrenewal of ESCs (Chambers et al., 2003; Darr et al., 2006), but only a limited amount of information is available on the upstream regulators that control the expression of this gene. Here, we describe a new function of retinol, the alcohol form of Vitamin A, in increasing the transcriptional output of Nanog by potentially a new signaling pathway, which is independent of the previously known LIF/Stat3, BMPs, Wnt/b-catenin, and Oct3/4-Sox2 pathways. Retinol and its metabolites are mainly associated with the differentiation during embryonic and fetal development (Clagett-Dame and DeLuca, 2002). With the exception of retinaldehyde, which is involved in vision (Wald, 1968), the regulation of cell functions by retinol are ascribed to retinoic acid (RA), a hormone well known for its role in differentiation of various cell types (Zile, 2001). Contrary to its reported functions, the studies described here demonstrate a new function of retinol in suppressing the differentiation of ESCs via the overexpression of Nanog. This function is independent of RA, suggesting the possibility of an alternate signaling pathway mediated by retinol or its yet to be identified metabolite/s. The studies offer a model system to obtain much needed information about the master regulators that are responsible for the ‘‘stemness’’ of ESCs.
Methods Embryonic stem cell culture Mouse ESCs were cultivated either feeder-free or on irradiated mouse embryonic fibroblasts (MEFs) in ES medium containing Dulbecco’s modified Eagle’s medium (DMEM) with 15% fetal bovine serum, 1 mM L-glutamine, 1% nonessential amino acids, 0.1 mM b-mercaptoethanol, and 1,000 U/ml LIF (Robertson, 1987). Normal or green florescent protein (GFP)-positive ESCs such as R1 cells, KBL2 cells, FVB/N13 cells from 129Sv, C57BL6, and FVB/N strains of mouse, respectively, were used for the studies. Stable GFP-positive cell lines were created by electroporation of pEGFP N1 vector (Clonetech, Mountain View, CA) followed by selection with G418. For feeder-free cultures, the confluent cultures of ESCs were trypsinized with 0.25% trypsin ethylenediaminetetraacetic acid (EDTA). The trypsinization was stopped by adding medium-containing fetal bovine serum followed by centrifugation. The pellet was resuspended in ES medium followed by transfer to 100 mm plates. After 1 hr, the nonadherent ESCs were recovered from the medium. For feeder-free cultures, low density (approximately 2 103 ) cells were cultured over six-well Petri dishes coated with 0.1% gelatin.
Retinol treatment of ESCs Retinol purchased from Sigma-Aldrich, Co. (Cat. # R7632, St. Louis, MO) was dissolved in 100% ethanol as 100 mM stock solution. The ESCs were allowed to settle for 24 hr following which the medium was supplemented with 0.03–0.5 mM retinol. The medium was changed every day with fresh retinol. These experiments were performed at least three times.
Collection of cells and isolation of RNA The cells were collected after trypsinization with 0.25% trypsin EDTA. To remove feeder cells, the trypsinized cells were plated over 100 mm dishes for 1 hr as described above. The cells were lyzed in STAT 60 solution (TEL-TEST, Friendswood, TX) and total RNA was isolated following instructions from the manufacturer.
Semi-quantitative reverse transcription PCR Total RNA isolated from ESCs was converted into cDNA using oligo-dT and avian myeloblastosis virus (AMV) reverse transcriptase in a final volume of 20 ml using kit purchased from Invitrogen (Carlsbad, CA). PCR was carried out using 2 ml aliquots in a total reaction volume of 50 ml using specific primers. The PCR conditions used denaturation 941C 45 sec; extension 721C 2 min; annealing at temperature as specified for each primer pair for 23–30 cycles. Products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining using HPRT primers as control. The RT-PCR analysis was performed three times. The intensity of the bands was quantitated by Bio-Rad Fluor-S-MultiImager (Hercules, CA) using Quantity one-4.2.3 software.
RT-PCR primer sequences Id2 F 5 0 -ACGAGCAGCATGAAAGCCTTCAGT-30 , R 5 0 -TT AGCCACAGAGTACTTTGCTATC-30 ; HPRT F 5 0 -GTAATGAT CAGTCAACGGGGGAC-3 0 , R 5 0 -CCAGCAAGCTTGCAACC TTAACCA-3 0 ; Oct3/4 F 5 0 - GGCGTTCTCTTTGGAAAGG TGTTC-3 0 , R 5 0 - CTCGAACCACATCCTTCTCT-3 0 ; Stat3 F 5 0 ATGAAGAGTGCCTTCGTGGTGG-30 , R 5 0 - GGATTGATGCC CAAGCATTTGG-3 0 ; Nanog F 5 0 -AGGGTCTGCTACTGAGA
684 TGCTCTG-3 0 , R 5 0 -CAACCACTGGTTTTTCTGCCACCG-3 0 annealing temperature 551C. Id1 F 5 0 -CAGGATCATGAAGGTCGCCAGTGG-3 0 , R 5 0 -AG TGCGCCGCCTCAGCGACACAAGA-3 0 ; Id3 F- 5 0 -TCTCCAA CATGAAGGCGCTGAGCCC-3 0 , R 5 0 -GTCAGTGGCAAAAG CTCCTCTTGTCC-3 0 ; Rex1 F 5 0 - ATCCGGGATGAAAGTGA GATTAGC-3 0 , R 5 0 -CTTCAGCATTTCTTCCCTGCCTTTGC -3 0 ; CYP26 F 5 0 -TTCTGCAGATGAAGCGCAGG-3 0 , R 5 0 -TT TCGCTGCTTGTGCGAGGA-3 0 annealing temp 611C. Sox2 F 5 0 -GAGAGCAAGTACTGGCAAGACCG-3 0 , R 5 0 - TA TACATGGATTCTCGCCAGCC-3 0 ; FGF4 F 5 0 -AGCGAGGC GTGGTGAGCATCTT-3 0 , R 5 0 -TGGTCCGCCCGTTCTTACT GAG-3 0 annealing temperature 641C. Western blot analysis Anti-Stat3 (c-20), anti-Oct4, anti-Sox2, and anti-Nanog antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Total protein was extracted with lysis buffer (Sigma Cat. #R0278). Fifty micrograms protein was separated by 12% SDS-PAGE and transferred onto a nylon membrane (Bio-Rad). The membranes were incubated with antibodies to specific protein followed by incubation with peroxidase-conjugated goat antibody to mouse IgG or rabbit antibody to goat IgG (Santa Cruz Biotechnology), and developed with chemiluminescence reagent (Pierce, Rockford, IL). The protein level was measured by Bio-Rad Fluor-S-MultiImager using Quantity one-4.2.3 software. Immunostaining of ES cells For immunostaining, ESCs were fixed with 4% paraformaldehyde and permeabilized with cold methanol. After washing and fixing, nonspecific binding was blocked with 5% FCS in phosphatebuffered saline (PBS) for 1 hr at room temperature. ES cells were labeled with goat anti-mouse Nanog antibody (Santa-Cruz Biotechnology) and Texas Red-conjugated donkey anti-goat antibody was used as secondary antibody. Cells were examined by fluorescence microscopy using low power Leica (Allendale, NJ) microscope. Alkaline phosphatase assay Cells were fixed with 4% paraformaldehyde for 2 min at room temperature. Staining for alkaline phosphatase was performed using a kit from Chemicon (Temecula, CA), following protocols provided by the manufacturer. Transfection of small interference RNAs (siRNA) Chemically synthesized 20–25 nt mixed oct4 siRNAs were obtained from Santa Cruz Biotechnology. Transfection of siRNA into ESCs was carried out in a six-well plate using transfection reagent according to the manufacturer’s protocol (Santa Cruz Biotechnology). The medium was removed 6 hr after transfection followed by the addition of 3 ml fresh ES medium with 0.25 mM retinol. Total RNA and protein were extracted 16 hr posttransfection. Nonspecific siRNAs were used as control. Chimera formation To determine the colonization potential, retinol-treated cells were co-cultured with 2.5 days morulas (Khillan and Bao, 1997) isolated from C57BL6 mice. After overnight culture, the blastocysts were transferred to the uterine horns of 2.5 days pseudopregnant CD1 foster mothers and the embryos were collected at specific days. Embryos were also allowed to develop to term to obtain chimeric animals. To prepare chimeric animals for C57BL6 ESCs, approxi-
mately 15–20 KBL2 cells were microinjected into 3.5 days blastocysts. All animal procedures were carried out according to the University of Pittsburgh-approved IACUC protocols.
Results Retinol-mediated suppression of ESC differentiation Mouse ESCs require LIF and feeder cells for maintenance and self-renewal (Stewart et al., 1992). To further refine our search for the factors involved in maintaining the pluripotency of ESCs, R1 ESCs were cultured over primary fibroblast feeders in ES medium. The cells were then treated with increasing concentrations, that is, 0.03–0.5 mM of retinol with daily change of the medium using fresh retinol. Approximately 50% of the cells in untreated cultures differentiated within first 5 days as judged by the flat morphology of the colonies. The number of differentiated colonies increased to over 80% after 8 days. At day 12, the cells were stained for alkaline phosphatase (AP), a specific marker for undifferentiated cells. None of the colonies stained positive with AP in untreated cultures (Fig. 1, top left panel) whereas the addition of retinol suppressed the differentiation of ESCs in a concentration-dependent manner. The cells showed strong AP staining at 0.125–0.5 mM concentration (Fig. 1, bottom panels). Almost 100% of the colonies remained undifferentiated at these concentrations. Lower concentrations such as 0.03 and 0.06 mM (upper middle and right panels), however, failed to suppress ESC differentiation as observed by the decreased AP staining. To investigate, whether retinol effect is dependent upon the background of the cells, ESCs derived from three independent mouse strains, that is, R1 cells from 129Sv, KBL2 cells from C57BL6, and FVB/N13 from FVB/N strain of mouse, were cultured over feeder cells using ES medium with 0.25 mM retinol. The untreated cells from all the three strains differentiated almost completely within 8 days. However, over 95% of the colonies in retinol-treated cells remained undifferentiated after 12 days (not shown) indicating that retinol can suppress the differentiation of ESCs irrespective of the strain background. Retinol-mediated suppression of ESC differentiation is independent of LIF Mouse ESCs are dependent upon LIF for their selfrenewal. Feeder cells support the self-renewal of ESCs by supplying LIF (Stewart et al. 1992). To investigate whether retinol plays any role in LIF/Stat3 pathway, single cell preparations of R1 ESCs were seeded at low density (about 2 103 cells in six-well plates) on feederfree gelatin-coated plates in the absence or presence of LIF (Figs. 2A, 2B, respectively). The cells were then
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Fig. 1 Retinol suppresses the differentiation of embryonic stem cells (ESCs) in long-term cultures. R1 ESCs were cultured over mouse embryo fibroblast feeder cells in embryonic stem cell medium. After 24 hr, the medium was supplemented with 0.03–0.5 mM
retinol. The medium was changed everyday with fresh retinol. The cells were stained for alkaline phosphatase (AP) after 12 days. Almost 100% cells show strong AP staining at 0.125–0.5 mM retinol concentration (bottom panels).
treated with various concentrations of retinol followed by staining for AP after 12 days. In the absence of LIF, the untreated cells formed flat and differentiated colonies within first 3–4 days and were completely differentiated by day 6 (Fig. 2A, top left panel), whereas 0.25– 0.5 mM retinol effectively suppressed the ESC differentiation up to 12 days (Fig. 2A, bottom panels). Over 60% of the colonies displayed typical ESC morphology and showed strong AP staining (bottom panels marked by arrow heads). Addition of LIF to these cultures had a strong positive effect on the morphology and growth of the cells as observed by the compact and well-formed colonies (Fig. 2B). Over 90% of the colonies remained undifferentiated at 0.25 and 0.5 mM retinol (Fig. 2B, bottom panels). The apparent low number of colonies in these cultures is firstly because the cultures were started with very low cell number (2 103 cells each well) and second in the absence of feeder cell many of the colonies were detached from the surface during staining process. These data demonstrate that although the suppression of differentiation is independent of LIF/Stat3 pathway, LIF has some cooperative effect with retinol for maintaining the undifferentiated growth of ESCs. No difference in cell number was observed in treated and untreated cultures from all the cell lines indicating that retinol does not provide any growth advantage to ESCs.
transcriptase polymerase chain reaction (RT–PCR). As shown in Figure 3A, retinol increased the expression of pluripotency promoting genes such as nanog, oct3/4, sox2, and rex1 in a concentration-dependent manner. Quantitation of amplified DNA fragments by BioRad digital densitometry analysis using Quantity one-4.2.3 software showed three-, two-, and fourfold increase in nanog, oct3/4, and sox2 message, respectively. Rex1, which is a downstream target of Nanog also showed twofold increase in its message. No change was observed in stat3 message and the fgf4 message, which is expressed in ICM of the blastocyst (Yuan et al., 1995). Similarly id genes such as id1, id2, and id3, whose expression is regulated by BMP signaling to maintain the pluripotency, also remained unchanged. Western blot analysis using antibodies against phosphorylated b-catenin, the active form, showed no change in b-catenin after retinol treatment (not shown). These RT-PCR data on various genes suggest that retinol-mediated suppression of ESC differentiation may occur via increased expression of nanog, oct3/4, and sox2 genes.
Relationship of retinol and the known regulators of ESC self-renewal To investigate the mechanisms by which retinol prevents ES cell differentiation, total RNA from retinoltreated cells was analyzed by semiquantitative reverse
Retinol induced overexpression of Nanog To investigate whether the message of these genes results in the protein synthesis, Western blot analysis was performed on the total proteins isolated from the ESCs treated with retinol for 24 hr. Western blot analysis revealed a concentration-dependent increase in Nanog synthesis (Fig. 3B) similar to its messenger RNA (Fig. 3A). The level of Nanog peaked to about three-fold at 0.125 mM and remained steady at higher concentrations such as 0.25 and 0.5 mM. On the other hand, inspite of increase in message, the levels of Sox2 and Oct3/4
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Fig. 2 The suppression of differentiation by retinol is independent of leukemia inhibitory factor (LIF). (A) Low-density R1 embryonic stem cells (ESCs) (approximately 2 103 cells) in each well of sixwell plates were seeded on gelatinized plates in embryonic stem (ES) medium without LIF. After 24 hr, the medium was supplemented with 0.03–0.5 mM retinol. The cells were stained for alkaline phosphatase after 12 days. Most colonies showed strong alkaline phos-
phatase staining at 0.25 and 0.5 mM retinol (marked by arrows). (B) Low-density R1 ESCs were seeded on gelatinized six-well plates using ES medium with LIF. After 24 hr, the medium was supplemented with 0.03–0.5 mM retinol. The cells were stained for alkaline phosphatase after 12 days. The retinol at 0.125–0.5 mM concentration effectively prevented the differentiation of ESCs even after 12 days of culture.
remained unchanged (Figs. 3B, 3C) suggesting a control of these genes at translational level. Also, no change was observed in the Stat3 protein (Fig. 3C) further supporting that the effect of retinol is independent of LIF/Stat3 pathway. Based on the earlier reports on overexpression of Nanog by Chambers et al. (2003) and Darr et al. (2006), these data strongly suggest that the suppression of ESC differentiation by retinol may be caused by the overexpression of Nanog.
Retinol-mediated overexpression of Nanog is independent of Oct3/4 and Sox2 The Nanog promoter contains binding sites for both positive and negative regulatory transcription factors including a pair of highly conserved Oct3/4-Sox2 factors (Kuroda et al., 2005; Rodda et al., 2005), a pluripotent cell-specific Sox element binding protein (PSBP) (Kuroda et al. 2005), Tcf3 (Pereira et al.,
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2006), and p53 (Lin et al., 2005). The overexpression of Nanog, therefore, can occur due to more efficient interactions of positive regulators such as Oct3/4-Sox2 (Fig. 4A, panel I) at the nanog promoter. Alternatively, retinol may induce the expression of yet unknown transcription factor/s that interact with nanog promoter either alone (Fig. 4A, panel II) or with Oct3/4-Sox2. To characterize such interactions, (siRNA) for oct3/4 gene were introduced into ESCs using lipofectamine. In spite of over 50% decrease in oct3/4 message, there was no change in the nanog mRNA (Fig. 4B, middle lanes). Whereas, the addition of retinol caused over twofold increase in the nanog message (right lanes) indicating that Nanog overexpression is independent of Oct3/4. The data was further confirmed by Western blot analysis that showed that in spite of approx. 60% decrease in Oct3/4 protein, no effect was observed on Nanog overexpression (Fig. 4C, right lanes). Time course of Nanog activation
Fig. 3 Retinol induced the expression of pluripotent cell-specific genes. R1 embryonic stem cells (ESCs) cultured over mouse embryo fibroblast feeders were treated with different concentrations of retinol using embryonic stem medium. After 24 hr, the ESCs were harvested by trypsinization followed by the separation of feeder cells. (A) Total RNA was analyzed for different genes by reverse transcriptase-polymerase chain reaction using primers specific for the undifferentiated cells and using primers for hypoxanthine guanine phosphorybosyl transferase (hprt) as control. The density of the amplified fragments was quantitated by Bio-Rad Fluor-S-MultiImager and Quantity one-4.2.3 software. (B) Total proteins were analyzed by Western blot analysis using antibodies specific for Nanog and Sox2. (C) Western blot analysis of total proteins using antibodies specific for Oct3/4 and Stat3 using b-Actin as control. The proteins were visualized by Chemi-luminescence and quantitated using Bio-Rad Fluor-S-MultiImager and Quantity one-4.2.3 software. Retinol caused the concentration-dependent increase in only Nanog.
In order to study the earliest effect of retinol on Nanog synthesis, total RNA from ESCs cultured with 0.25 mM retinol for 15, 30, 60, 90, and 120 min was analyzed by RT-PCR analysis. There was a twofold increase in nanog message within the first 60 min of the treatment that increased only slightly after 2 hr, whereas no change was observed in the oct3/4 or stat3 message (Fig. 5A). The Western blot analysis of the same samples revealed a twofold increase in Nanog within first 60 min of the treatment (Fig. 5B). The level peaked to about threefolds in 4 hr and remained steady thereafter. This leads to the conclusion that retinal-induced signaling is highly efficient in up-regulating the nanog expression and the activation of Nanog occurs shortly after the treatment. To prove the expression of Nanog at cellular level, the retinol-treated cells were immunostained using Nanog-specific antibodies. Figure 5C shows that 5-day retinol treatment caused a dramatic increase in Nanog expression at 0.25 and 0.5 mM concentration (panels IV and VI, respectively) as compared with untreated cells (panel II). Enhanced-expression of Nanog is not mediated through retinoic acid Normally, the retinol is oxidized to retinaldehyde in cells by alcohol dehydrogenase 4 (ADH4), which is then oxidized to RA by retinaldehyde dehydrogenase (RALDH-2) (Zhao et al., 1996). ADH4, however, is not detectable until E6.5 during development (Ang and Duester, 1997). RA receptors (retinoic acid receptors [RARs] and retinoid X receptors [RXRs]) are expressed in ESCs and throughout the developing embryo (Hofmann and Eichek, 1994; Chen and Gudas, 1996; Ross et al., 2000). To investigate whether the effect of retinol
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Fig. 4 Overexpression of Nanog by retinol is independent of Oct3/ 4. (A) As nanog promoter contains Oct3/4 and Sox2 binding sites, retinol may induce certain factors that may cause more efficient binding of Sox2-Oct3/4 to nanog promoter (I) or the retinol-induced factors may bind directly to the nanog promoter (II). (B) oct3/4 siRNAs were introduced into ESCs. Reverse transcriptasepolymerase chain reaction analysis of RNA from cells treated with
retinol for 10 hr (Si1Re) and untreated cells (Si). Normal-untreated cells were used as control. (C) Western blot analysis on total proteins isolated from the same cells, treated with retinol for 10 hr (Si1Re) and untreated cells (Si) using antibodies specific for Oct3/4 and Nanog. The overexpression of Nanog protein and mRNA is obvious in spite of decrease in OC3/4. Non-specific siRNAs were used as control for both the experiments.
on Nanog occurs via RA-mediated signaling mechanism; ESCs were treated with 0.25 mM retinol and 1.0 mM RA. After 5 days the cells were tested for alkaline phosphate staining. As shown in Figure 6 (panel III), the retinoic acid caused complete differentiation of ES cells within 5 days. On the other hand, 100% of the colonies remained undifferentiated after treatment with retinol (panel II), whereas over 70% of the cells differentiated when the retinol was supplemented with 1 mM retinoic acid (panel IV) providing indirect evidence that retinol is not metabolized to RA. This also proves that RA and retinol follow independent pathways in ESCs. Furthermore, it is also clear from the data that retinol is unable to counter the RA-mediated differentiation.
were then transferred to the pseudopregnant mothers and the embryos were recovered at different stages such as E7.5 and E16.0 (mid-gestation period). The embryos were then visualized under UV light for ESC integration. Retinol-treated ESCs retained complete potential to contribute to the embryo and extra-embryonic membranes (Fig. 7A, top right panel) and showed high penetration in all the organs (bottom right panel). Further, the cells were also capable of generating live chimeras (Fig. 7B), confirming that retinol does not impair ESC potential for contributing to the different tissues of the embryo. Similarly KBL2 ESCs derived from C57BL6 embryo also produced highly chimeric animals when injected into FVB/N (albino) embryos after treatment with retinol (Fig. 7C).
Retinol does not impair embryo colonization properties of ESCs Pluripotent ESCs have the complete potential to colonize tissues derived from all the primary germ layers. To investigate whether retinol affects their ability to contribute to the various tissues, R1 ESCs were prepared that contained a gene for enhanced green florescence protein (EGFP) stably integrated into their genome (Khillan J. S., unpublished results). The cells were propagated for five passages in the medium supplemented with 0.25 mM retinol followed by aggregation with 2.5 days morulas (Khillan and Bao, 1997). The blastocysts developed from the aggregated embryos
Discussion To sustain their self-renewal, ESCs require a circuitry of different signaling pathways including LIF/Stat3 pathway, BMPs mediated activation of Ids, Wnt/b-catenin signaling, POU-domain-containing oct3/4, and homeodomain-containing sox2 and nanog genes (Smith, 2001). Both Oct3/4 and Nanog are expressed in the early stages in the ICM and the epiblast of the developing embryo. Gene knock-out studies in mouse have demonstrated that Oct3/4 and Nanog are essential for selfrenewal of ESCs (Nichols et al., 1998; Mitsui et al.,
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Fig. 5 Rapid induction of Nanog expression by retinol. Embroynic stem cells cultured over the feeder cells were treated with 0.25 mM retinol for 15min–6 hr. At each interval the cells were treated with lysis buffer. (A) Total RNA isolated from the cells was amplified by reverse transcriptase-polymerase chain reaction. A significant increase in nanog message was observed in 60 min of treatment. (B) Western blot analysis on total proteins from the cells treated with 0.25 mM retinol for 30 min to 6 hr. At each specific time point, the cells were analyzed using Nanog-specific antibodies. Approximately two-fold increase in Nanog was seen at protein level after 1 hr treatment with retinol. (C) Immunostaining of retinol-treated cells. Embryonic stem cells were cultured with 0.25 and 0.5 mM retinol for 5 days. The cells were then immunostained using antibodies against Nanog. A strong Nanog expression was observed in the cells treated with retinol (panels IV and VI) compared with the untreated cells (panel II).
2003). In the developmental hierarchy, nanog is expressed after oct3/4 (Pesce and Scholer, 2001). The first sign of nanog message is seen in the compacted morulae. Inactivation of oct3/4 results in the differentiation of ICM into trophectoderm (Nichols et al., 1998) whereas
nanog inactivation results in the ICM that fails to form epiblast and produces only parietal endoderm-like cells (Mitsui et al., 2003). Although the nanog expression is unaffected by the absence of oct3/4, a sustained expression of oct3/4 is essential for self-renewal of ESCs. The addition of LIF, however, can have an enhancing effect on their growth (Chambers et al., 2003). Recently, Chambers et al. (2003) and Darr et al. (2006) have shown that overexpression of Nanog can promote the self-renewal of ESCs thus indicating that it is important for maintaining the pluripotency. However, no effect on the cell proliferation was reported. Our studies have shown that addition of retinol to the culture medium did not increase the proliferation of ESCs as the cell number between treated and untreated cultures remained almost same in spite of threefold overexpression of Nanog even after six passages (data not shown). Recently, Zhang et al. (2005) reported that expression of Nanog promotes the proliferation of NIH 3T3 cells. Using three independent cell lines such as Cos 7, HEK293, and NIH3T3, Peistun et al. (2006), on the other hand, reported that Nanog can transform NIH3T3 cells. They further showed that Nanog targets specific set of genes that is unique to each cell type. In this respect, the overexpression of Nanog in ESCs may only be responsible for maintaining the optimal level of pluripotency-specific genes. This phenomenon needs further investigation. Overexpression of Nanog without any effect on Stat3 supports the earlier observations that Nanog can bypass the LIF/Stat3 pathway, though a certain level of Nanog is critical for maintaining the cytokine-independent selfrenewal (Chambers et al., 2003). A threefold increase in Nanog by retinol, therefore, appears to satisfy that requirement. However, further studies are needed to prove this. Under normal conditions, the ESC cultures require trypsinization every 3–4 days to break up the colonies to prevent differentiation. Retinol, on the other hand, supported the growth of undifferentiated cells for 12 days. The effect of retinol beyond 12 days was not tested. Further analysis of cells by RT-PCR for differentiation-specific genes fgf8, nestin, and brachyury did not show amplification of the specific fragments suggesting the undifferentiated nature of the cells. Although the ESCs exhibited a dose-dependent response, the net effect of retinol remained the activation of Nanog. In all the cultures described here, the retinol concentrations used were less than the physiological levels (1.0–2.0 mM) (Lane et al., 1999). A retinol concentration as high as 2.0 mM also did not show any obvious toxic effect on ESCs; however, the cells started to bleb after one to two passages. Therefore, for all the subsequent analyses we used only 0.25 mM concentration, which was sufficient to maintain the pluripotency of ESCs. Nanog function is conserved between species; for example human NANOG can direct LIF independent
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Fig. 6 Suppression of Embryonic stem cells (ESC) differentiation by retinol does not involve signaling by retinoic acid. ESCs cultured over mouse embryo fibroblast feeder cells were treated with 0.25 mM retinol in the absence (Re) and presence of 1.0 mM retinoic acid (RA) (Re1RA). Fresh medium was used everyday with
fresh retinol and retinoic acid. The cells were stained for alkaline phosphatase after 5 days. Addition of retinoic acid caused differentiation of ESCs in the presence (panel IV) or absence (panel III) of retinol. Almost all the cells remained undifferentiated in retinolcontaining medium (panel II). Control cells (panel I).
self-renewal of mouse and monkey ESCs (Chambers et al., 2003; Yasuda et al., 2006). Mouse Nanog has 87% and 58% identity with rat and human orthologs, respectively. This sequence identity is more pronounced in the homeodomain region (94% for rat and 87% for humans) (Chambers et al., 2003). Nanog may sustain the pluripotency of ESCs either by activating the undifferentiated cell-specific genes and/or by the suppression of differentiation-promoting genes chromatin immuno precipitation (ChIP)- pair end ditag (PET) assay revealed many genes that contain Nanog binding sites (Loh et al., 2006). Differentiation-specific genes such as gata4 and gata6 contain DNA recognition motif of Nanog and their expression is up-regulated in Nanog null cells (Mitsui et al., 2003). Our data clearly demonstrate that the retinol-mediated activation of nanog is independent of known signaling pathways including Jak/Stat3 pathway, BMP/Id, Wnt/b-catenin signaling, and Oct3/4-Sox2 suggesting the possibility of an alternate signaling mechanism by retinol. Though the binding of Oct3/4-Sox2 at the conserved binding sites in the nanog promoter have been shown to regulate its expression (Kuroda et al., 2005; Rodda et al., 2005), our RNAi data suggest that retinol effect may not occur via this mechanism and may involve some unknown transcription factors. Further, in spite of increases in their message, the Oct3/4 and Sox2 levels remained unchanged suggesting a control at the translation level. In this respect it is worth noting the studies described by Kuroda et al. (2005) where they demonstrated that the expression of a reporter gene from nanog promoter is 15% higher when 2.5 kb upstream region was used as compared with Oct3/4Sox2 binding sites containing 353 bp region indicat-
ing the involvement of other factors in nanog regulation. Our studies, however, do not completely rule out the role of Oct3/4-Sox2 as after repeated attempts we were unable to eliminate the oct3/4 message. On the other hand, the absence of any change in fgf4 levels, a gene that also contains binding sites for Oct3/4 and Sox 2 (Kuroda et al., 2005), lends support for Oct3/4- and Sox2-independent mechanism. Retinol-mediated overexpression may occur either due to the interaction of some unknown transcription factor/s with Oct3/4-Sox2 (Fig. 4, panel I) or alternatively via binding of this factor/s to some specific regulatory elements in the nanog promoter (panel II). The studies are currently in progress to identify such a potential element/s in the nanog promoter. However, with the available data we cannot completely rule out the role of these two factors. Further, we have not yet tested the auto-regulation by Nanog for which we are generating gene constructs with different promoter lengths and reporter luciferase gene. Retinol, the alcohol form of vitamin A, and its derivatives play an essential role in many biological functions during development of the embryo. Thus far no specific biological role has been assigned to retinol as such. With the exception of retinaldehyde, which is involved in visual cycle (Wald, 1968), the biologically active form of retinol is RA that regulates many differentiation specific genes by binding to the RAR and RXR (Balmer and Blomhoff, 2002; Clagett-Dame and DeLuca, 2002). A notable example of ESC differentiation by retinol metabolites was reported by Lane et al., (1999). A careful review of these studies revealed a critical difference in the methodology to culture the cells that affected the outcome of their investigations. These
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Fig. 7 Retinol does not affect the integration potential of embryonic stem cells (ESCs) into different tissues. Green florescence protein (EGFP)-positive R1 ESCs and C57BL6 ESCs were cultured in the presence of 0.25 mM retinol for five passages. (A) C57BL6 embryos were microinjected with R1ESCs and the embryos were recovered from mothers at E7.5 and E16.0. The embryos and fetuses
were visualized under UV light. An arrow in the top left panel shows the direction of the uterine attachment. (B) Chimeric animal generated by aggregation of C57BL6 morula and R1ESCs. (C) Chimeric pups generated by microinjection of C57BL6 ESCs into FVB/N (albino) blastocysts.
investigators cultured ESCs without feeders and LIF for 96 hr before the addition of retinol. Under such conditions, the ESCs are expected to be already in the state of differentiation. In the current studies, the retinol was added almost immediately after the cells had settled down. Our data also rule out the possibility of involvement of RA that suggests a provocative hypothesis that the suppression of differentiation in ESCs may be caused by some novel signaling mechanism mediated via yet unknown retinol metabolite/s. Analysis of the retinoltreated ESCs by HPLC analysis may reveal the identity of such molecules. The alternate pathway used by retinol to suppress differentiation may be important to maintain the functional balance of retinol during embryonic and fetal development. The regulation of nanog by a physiologically relevant compound such as retinol offers a unique opportunity
to identify transcription factors, which control its expression that may lead to much needed information about the master regulatory genes responsible for the ‘‘stemness’’ of the cell. Colonization of retinol-treated ESCs into embryos, extra-embryonic membranes, and chimeric animal proves that retinol does not affect the pluripotency of ESCs. Until now, very little information is available on the in vivo and in vitro role of retinol on embryo development. Recently, Livingston et al. (2004) demonstrated that retinol can improve the development of bovine embryos in vitro. Studies are currently in progress in our lab to investigate the in vitro effect of retinol on preimplantation embryo as well as post-implantation embryo in vivo after transfer of retinol-treated embryos into pseudopregnant mothers. Overexpression of Nanog by retinol may be of great significance for the development
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and stabilization of new ES cell lines from human embryos especially from the embryos generated for therapeutic cloning after somatic cell nuclear transfer. Studies are in progress to investigate whether the human NANOG also responds to retinol in a similar fashion. Our preliminary studies have shown that human ESCs form compact colonies following retinol treatment.
Acknowledgments We thank Dr. Arthur S. Levine, Dean of Graduate Studies, University of Pittsburgh, for providing the support for these studies and Drs. Devjani Chatterjee and Sanjay Mishra for critical reading of the manuscript. We also thank Dr. P. Monga for providing antibodies for b-catenin.
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OR IGI N A L A R T IC L E
Liguo Chen . Minying Yang . Joyce Dawes . Jaspal Singh Khillan
Suppression of ES cell differentiation by retinol (vitamin A) via the overexpression of Nanog
Received November 13, 2006; accepted in revised form January 4, 2007
Abstract Embryonic stem cells (ESCs) derived from the inner cell mass of blastocysts maintain their pluripotency through a complex interplay of different signaling pathways and transcription factors including Leukemia Inhibitory Factor (LIF), homeo-domain protein Nanog and POU-domain-containing transcription factor Oct3/4. LIF can maintain the self-renewal of mouse ESCs by activating the Jak/Stat3 pathway; however, it is dispensable for human ESCs. Nanog, a homeo-domain transcription factor alone is sufficient for sustaining the self-renewal of ESCs. Overexpression of Nanog by heterologous promoters can maintain selfrenewal of human and mouse ESCs in the absence of LIF/Stat3 pathway. The mechanisms that control the expression of Nanog, however, remain poorly understood. In this report we demonstrate that retinol, the alcohol form of Vitamin A, can suppress the differentiation of ESCs by up-regulating the expression of Nanog. Retinol is mainly associated with differentiation through its active metabolite retinoic acid during early development of the embryo. The activation of Nanog by retinol is not mediated via retinoic acid signaling and appears to be independent of previously described LIF/ Stat3, bone morphogenic proteins, Wnt/b-catenin, and Oct3/4-Sox2 pathways. These studies therefore, reveal a previously unknown function of retinol and offer a model system to define alternate regulatory pathways that control the self-renewal of ESCs as well as to identify upstream ‘‘master’’ regulatory factors that are responsible for maintaining the integrity of stem cells.
Liguo Chen Minying Yang Joyce Dawes Jaspal Singh . ) Khillan (* Department of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. Tel: 11 412 383 6987 Fax: 11 412 865 8695 E-mail: [email protected]
Key words self renewal of ES cells overexpression of Nanog retinol (Vitamin A) retinoic acid suppression of ES cell differentiation Oct3/4
Introduction Embryonic stem cells (ESCs) derived from the inner cell mass (ICM) of mammalian blastocysts have the potential to grow indefinitely while maintaining pluripotency. After injection into blastocysts, ESCs can contribute to all the primary germ layers such as endoderm, mesoderm, and ectoderm (Evans and Kaufman, 1981; Martin, 1981). Depending upon the culture conditions, these cells can differentiate into different cell types (Smith, 2001). Because of these properties, ESCs will hopefully be useful in treating degenerative diseases such as Parkinson’s disease, spinal cord injury, diabetes, and cardiovascular diseases in humans (Thomson et al., 1998). ESCs maintain pluripotency by expressing a specific transcriptional program that suppresses their differentiation. Leukemia inhibitory factor (LIF), a member of IL6 family of lymphokines, can maintain the self-renewal of ESCs by activating Stat3 (Smith et al., 1988; Williams et al., 1988; Stewart et al., 1992; Smith and Crompton, 1998). LIF binds to a heterodimer receptor, consisting of LIFR and GP130 that lead to the activation of Jak/Stat3 pathway (Ernst et al., 1996; Ihle et al., 1996). This pathway then activates the downstream genes (Davis et al., 1994). Although LIF signaling is important for mouse ESCs (Burdon et al., 2002), it is not sufficient to sustain their self-renewal and requires additional signals. These signals are provided by the bone morphogenic proteins (BMPs), which activate inhibitors of differentiation (Id) genes (Ying et al., 2003). LIF/Stat3 signaling, however, is dispensable for human ESCs (Daheron et al., 2004) suggesting that this pathway is not fundamental for maintenance of pluripoten-
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cy. Furthermore, Wnt/b-catenin signaling has also been implicated in maintaining the undifferentiated status of both human and mouse ESCs (Saito et al., 2004). POU domain-containing transcriptional factor Oct3/ 4, homeo-box genes nanog and sox2 and fibroblast growth factor 4 (fgf4) play a key role in maintaining the pluripotency of ESCs (Yuan et al., 1995; Wilder et al., 1997; Nichols et al., 1998; Avilion et al., 2003; Chambers et al., 2003; Mitsui et al., 2003). The oct3/4 and nanog are specifically expressed in ICM and epiblast of the early embryo (Nichols et al., 1998; Avilion et al., 2003; Chambers et al., 2003; Mitsui et al., 2003). Null mutations of oct3/4 (Nichols et al., 1998) and nanog (Mitsui et al., 2003) result in early embryonic lethality. Nanog alone, however, can maintain the self-renewal and pluripotency of ESCs independent of LIF/Stat3 pathway (Chambers et al., 2003; Mitsui et al., 2003). Down-regulation of Nanog results in the differentiation of ESCs into extra embryonic endoderm, whereas overexpression of Nanog allows propagation and self-renewal of ESCs in the absence of LIF (Darr et al., 2006). Although many signaling pathways are involved in the self-renewal of ESCs (Smith, 2001), only limited information is available about the regulatory mechanisms that control the key genes responsible. An understanding of the circuitary of transcription factors that maintains the pluripotency of ESCs is fundamental to realizing their full potential for therapeutic applications. The overexpression of Nanog by heterologous promoters has been used as a model to study the selfrenewal of ESCs (Chambers et al., 2003; Darr et al., 2006), but only a limited amount of information is available on the upstream regulators that control the expression of this gene. Here, we describe a new function of retinol, the alcohol form of Vitamin A, in increasing the transcriptional output of Nanog by potentially a new signaling pathway, which is independent of the previously known LIF/Stat3, BMPs, Wnt/b-catenin, and Oct3/4-Sox2 pathways. Retinol and its metabolites are mainly associated with the differentiation during embryonic and fetal development (Clagett-Dame and DeLuca, 2002). With the exception of retinaldehyde, which is involved in vision (Wald, 1968), the regulation of cell functions by retinol are ascribed to retinoic acid (RA), a hormone well known for its role in differentiation of various cell types (Zile, 2001). Contrary to its reported functions, the studies described here demonstrate a new function of retinol in suppressing the differentiation of ESCs via the overexpression of Nanog. This function is independent of RA, suggesting the possibility of an alternate signaling pathway mediated by retinol or its yet to be identified metabolite/s. The studies offer a model system to obtain much needed information about the master regulators that are responsible for the ‘‘stemness’’ of ESCs.
Methods Embryonic stem cell culture Mouse ESCs were cultivated either feeder-free or on irradiated mouse embryonic fibroblasts (MEFs) in ES medium containing Dulbecco’s modified Eagle’s medium (DMEM) with 15% fetal bovine serum, 1 mM L-glutamine, 1% nonessential amino acids, 0.1 mM b-mercaptoethanol, and 1,000 U/ml LIF (Robertson, 1987). Normal or green florescent protein (GFP)-positive ESCs such as R1 cells, KBL2 cells, FVB/N13 cells from 129Sv, C57BL6, and FVB/N strains of mouse, respectively, were used for the studies. Stable GFP-positive cell lines were created by electroporation of pEGFP N1 vector (Clonetech, Mountain View, CA) followed by selection with G418. For feeder-free cultures, the confluent cultures of ESCs were trypsinized with 0.25% trypsin ethylenediaminetetraacetic acid (EDTA). The trypsinization was stopped by adding medium-containing fetal bovine serum followed by centrifugation. The pellet was resuspended in ES medium followed by transfer to 100 mm plates. After 1 hr, the nonadherent ESCs were recovered from the medium. For feeder-free cultures, low density (approximately 2 103 ) cells were cultured over six-well Petri dishes coated with 0.1% gelatin.
Retinol treatment of ESCs Retinol purchased from Sigma-Aldrich, Co. (Cat. # R7632, St. Louis, MO) was dissolved in 100% ethanol as 100 mM stock solution. The ESCs were allowed to settle for 24 hr following which the medium was supplemented with 0.03–0.5 mM retinol. The medium was changed every day with fresh retinol. These experiments were performed at least three times.
Collection of cells and isolation of RNA The cells were collected after trypsinization with 0.25% trypsin EDTA. To remove feeder cells, the trypsinized cells were plated over 100 mm dishes for 1 hr as described above. The cells were lyzed in STAT 60 solution (TEL-TEST, Friendswood, TX) and total RNA was isolated following instructions from the manufacturer.
Semi-quantitative reverse transcription PCR Total RNA isolated from ESCs was converted into cDNA using oligo-dT and avian myeloblastosis virus (AMV) reverse transcriptase in a final volume of 20 ml using kit purchased from Invitrogen (Carlsbad, CA). PCR was carried out using 2 ml aliquots in a total reaction volume of 50 ml using specific primers. The PCR conditions used denaturation 941C 45 sec; extension 721C 2 min; annealing at temperature as specified for each primer pair for 23–30 cycles. Products were resolved by agarose gel electrophoresis and visualized by ethidium bromide staining using HPRT primers as control. The RT-PCR analysis was performed three times. The intensity of the bands was quantitated by Bio-Rad Fluor-S-MultiImager (Hercules, CA) using Quantity one-4.2.3 software.
RT-PCR primer sequences Id2 F 5 0 -ACGAGCAGCATGAAAGCCTTCAGT-30 , R 5 0 -TT AGCCACAGAGTACTTTGCTATC-30 ; HPRT F 5 0 -GTAATGAT CAGTCAACGGGGGAC-3 0 , R 5 0 -CCAGCAAGCTTGCAACC TTAACCA-3 0 ; Oct3/4 F 5 0 - GGCGTTCTCTTTGGAAAGG TGTTC-3 0 , R 5 0 - CTCGAACCACATCCTTCTCT-3 0 ; Stat3 F 5 0 ATGAAGAGTGCCTTCGTGGTGG-30 , R 5 0 - GGATTGATGCC CAAGCATTTGG-3 0 ; Nanog F 5 0 -AGGGTCTGCTACTGAGA
684 TGCTCTG-3 0 , R 5 0 -CAACCACTGGTTTTTCTGCCACCG-3 0 annealing temperature 551C. Id1 F 5 0 -CAGGATCATGAAGGTCGCCAGTGG-3 0 , R 5 0 -AG TGCGCCGCCTCAGCGACACAAGA-3 0 ; Id3 F- 5 0 -TCTCCAA CATGAAGGCGCTGAGCCC-3 0 , R 5 0 -GTCAGTGGCAAAAG CTCCTCTTGTCC-3 0 ; Rex1 F 5 0 - ATCCGGGATGAAAGTGA GATTAGC-3 0 , R 5 0 -CTTCAGCATTTCTTCCCTGCCTTTGC -3 0 ; CYP26 F 5 0 -TTCTGCAGATGAAGCGCAGG-3 0 , R 5 0 -TT TCGCTGCTTGTGCGAGGA-3 0 annealing temp 611C. Sox2 F 5 0 -GAGAGCAAGTACTGGCAAGACCG-3 0 , R 5 0 - TA TACATGGATTCTCGCCAGCC-3 0 ; FGF4 F 5 0 -AGCGAGGC GTGGTGAGCATCTT-3 0 , R 5 0 -TGGTCCGCCCGTTCTTACT GAG-3 0 annealing temperature 641C. Western blot analysis Anti-Stat3 (c-20), anti-Oct4, anti-Sox2, and anti-Nanog antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Total protein was extracted with lysis buffer (Sigma Cat. #R0278). Fifty micrograms protein was separated by 12% SDS-PAGE and transferred onto a nylon membrane (Bio-Rad). The membranes were incubated with antibodies to specific protein followed by incubation with peroxidase-conjugated goat antibody to mouse IgG or rabbit antibody to goat IgG (Santa Cruz Biotechnology), and developed with chemiluminescence reagent (Pierce, Rockford, IL). The protein level was measured by Bio-Rad Fluor-S-MultiImager using Quantity one-4.2.3 software. Immunostaining of ES cells For immunostaining, ESCs were fixed with 4% paraformaldehyde and permeabilized with cold methanol. After washing and fixing, nonspecific binding was blocked with 5% FCS in phosphatebuffered saline (PBS) for 1 hr at room temperature. ES cells were labeled with goat anti-mouse Nanog antibody (Santa-Cruz Biotechnology) and Texas Red-conjugated donkey anti-goat antibody was used as secondary antibody. Cells were examined by fluorescence microscopy using low power Leica (Allendale, NJ) microscope. Alkaline phosphatase assay Cells were fixed with 4% paraformaldehyde for 2 min at room temperature. Staining for alkaline phosphatase was performed using a kit from Chemicon (Temecula, CA), following protocols provided by the manufacturer. Transfection of small interference RNAs (siRNA) Chemically synthesized 20–25 nt mixed oct4 siRNAs were obtained from Santa Cruz Biotechnology. Transfection of siRNA into ESCs was carried out in a six-well plate using transfection reagent according to the manufacturer’s protocol (Santa Cruz Biotechnology). The medium was removed 6 hr after transfection followed by the addition of 3 ml fresh ES medium with 0.25 mM retinol. Total RNA and protein were extracted 16 hr posttransfection. Nonspecific siRNAs were used as control. Chimera formation To determine the colonization potential, retinol-treated cells were co-cultured with 2.5 days morulas (Khillan and Bao, 1997) isolated from C57BL6 mice. After overnight culture, the blastocysts were transferred to the uterine horns of 2.5 days pseudopregnant CD1 foster mothers and the embryos were collected at specific days. Embryos were also allowed to develop to term to obtain chimeric animals. To prepare chimeric animals for C57BL6 ESCs, approxi-
mately 15–20 KBL2 cells were microinjected into 3.5 days blastocysts. All animal procedures were carried out according to the University of Pittsburgh-approved IACUC protocols.
Results Retinol-mediated suppression of ESC differentiation Mouse ESCs require LIF and feeder cells for maintenance and self-renewal (Stewart et al., 1992). To further refine our search for the factors involved in maintaining the pluripotency of ESCs, R1 ESCs were cultured over primary fibroblast feeders in ES medium. The cells were then treated with increasing concentrations, that is, 0.03–0.5 mM of retinol with daily change of the medium using fresh retinol. Approximately 50% of the cells in untreated cultures differentiated within first 5 days as judged by the flat morphology of the colonies. The number of differentiated colonies increased to over 80% after 8 days. At day 12, the cells were stained for alkaline phosphatase (AP), a specific marker for undifferentiated cells. None of the colonies stained positive with AP in untreated cultures (Fig. 1, top left panel) whereas the addition of retinol suppressed the differentiation of ESCs in a concentration-dependent manner. The cells showed strong AP staining at 0.125–0.5 mM concentration (Fig. 1, bottom panels). Almost 100% of the colonies remained undifferentiated at these concentrations. Lower concentrations such as 0.03 and 0.06 mM (upper middle and right panels), however, failed to suppress ESC differentiation as observed by the decreased AP staining. To investigate, whether retinol effect is dependent upon the background of the cells, ESCs derived from three independent mouse strains, that is, R1 cells from 129Sv, KBL2 cells from C57BL6, and FVB/N13 from FVB/N strain of mouse, were cultured over feeder cells using ES medium with 0.25 mM retinol. The untreated cells from all the three strains differentiated almost completely within 8 days. However, over 95% of the colonies in retinol-treated cells remained undifferentiated after 12 days (not shown) indicating that retinol can suppress the differentiation of ESCs irrespective of the strain background. Retinol-mediated suppression of ESC differentiation is independent of LIF Mouse ESCs are dependent upon LIF for their selfrenewal. Feeder cells support the self-renewal of ESCs by supplying LIF (Stewart et al. 1992). To investigate whether retinol plays any role in LIF/Stat3 pathway, single cell preparations of R1 ESCs were seeded at low density (about 2 103 cells in six-well plates) on feederfree gelatin-coated plates in the absence or presence of LIF (Figs. 2A, 2B, respectively). The cells were then
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Fig. 1 Retinol suppresses the differentiation of embryonic stem cells (ESCs) in long-term cultures. R1 ESCs were cultured over mouse embryo fibroblast feeder cells in embryonic stem cell medium. After 24 hr, the medium was supplemented with 0.03–0.5 mM
retinol. The medium was changed everyday with fresh retinol. The cells were stained for alkaline phosphatase (AP) after 12 days. Almost 100% cells show strong AP staining at 0.125–0.5 mM retinol concentration (bottom panels).
treated with various concentrations of retinol followed by staining for AP after 12 days. In the absence of LIF, the untreated cells formed flat and differentiated colonies within first 3–4 days and were completely differentiated by day 6 (Fig. 2A, top left panel), whereas 0.25– 0.5 mM retinol effectively suppressed the ESC differentiation up to 12 days (Fig. 2A, bottom panels). Over 60% of the colonies displayed typical ESC morphology and showed strong AP staining (bottom panels marked by arrow heads). Addition of LIF to these cultures had a strong positive effect on the morphology and growth of the cells as observed by the compact and well-formed colonies (Fig. 2B). Over 90% of the colonies remained undifferentiated at 0.25 and 0.5 mM retinol (Fig. 2B, bottom panels). The apparent low number of colonies in these cultures is firstly because the cultures were started with very low cell number (2 103 cells each well) and second in the absence of feeder cell many of the colonies were detached from the surface during staining process. These data demonstrate that although the suppression of differentiation is independent of LIF/Stat3 pathway, LIF has some cooperative effect with retinol for maintaining the undifferentiated growth of ESCs. No difference in cell number was observed in treated and untreated cultures from all the cell lines indicating that retinol does not provide any growth advantage to ESCs.
transcriptase polymerase chain reaction (RT–PCR). As shown in Figure 3A, retinol increased the expression of pluripotency promoting genes such as nanog, oct3/4, sox2, and rex1 in a concentration-dependent manner. Quantitation of amplified DNA fragments by BioRad digital densitometry analysis using Quantity one-4.2.3 software showed three-, two-, and fourfold increase in nanog, oct3/4, and sox2 message, respectively. Rex1, which is a downstream target of Nanog also showed twofold increase in its message. No change was observed in stat3 message and the fgf4 message, which is expressed in ICM of the blastocyst (Yuan et al., 1995). Similarly id genes such as id1, id2, and id3, whose expression is regulated by BMP signaling to maintain the pluripotency, also remained unchanged. Western blot analysis using antibodies against phosphorylated b-catenin, the active form, showed no change in b-catenin after retinol treatment (not shown). These RT-PCR data on various genes suggest that retinol-mediated suppression of ESC differentiation may occur via increased expression of nanog, oct3/4, and sox2 genes.
Relationship of retinol and the known regulators of ESC self-renewal To investigate the mechanisms by which retinol prevents ES cell differentiation, total RNA from retinoltreated cells was analyzed by semiquantitative reverse
Retinol induced overexpression of Nanog To investigate whether the message of these genes results in the protein synthesis, Western blot analysis was performed on the total proteins isolated from the ESCs treated with retinol for 24 hr. Western blot analysis revealed a concentration-dependent increase in Nanog synthesis (Fig. 3B) similar to its messenger RNA (Fig. 3A). The level of Nanog peaked to about three-fold at 0.125 mM and remained steady at higher concentrations such as 0.25 and 0.5 mM. On the other hand, inspite of increase in message, the levels of Sox2 and Oct3/4
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Fig. 2 The suppression of differentiation by retinol is independent of leukemia inhibitory factor (LIF). (A) Low-density R1 embryonic stem cells (ESCs) (approximately 2 103 cells) in each well of sixwell plates were seeded on gelatinized plates in embryonic stem (ES) medium without LIF. After 24 hr, the medium was supplemented with 0.03–0.5 mM retinol. The cells were stained for alkaline phosphatase after 12 days. Most colonies showed strong alkaline phos-
phatase staining at 0.25 and 0.5 mM retinol (marked by arrows). (B) Low-density R1 ESCs were seeded on gelatinized six-well plates using ES medium with LIF. After 24 hr, the medium was supplemented with 0.03–0.5 mM retinol. The cells were stained for alkaline phosphatase after 12 days. The retinol at 0.125–0.5 mM concentration effectively prevented the differentiation of ESCs even after 12 days of culture.
remained unchanged (Figs. 3B, 3C) suggesting a control of these genes at translational level. Also, no change was observed in the Stat3 protein (Fig. 3C) further supporting that the effect of retinol is independent of LIF/Stat3 pathway. Based on the earlier reports on overexpression of Nanog by Chambers et al. (2003) and Darr et al. (2006), these data strongly suggest that the suppression of ESC differentiation by retinol may be caused by the overexpression of Nanog.
Retinol-mediated overexpression of Nanog is independent of Oct3/4 and Sox2 The Nanog promoter contains binding sites for both positive and negative regulatory transcription factors including a pair of highly conserved Oct3/4-Sox2 factors (Kuroda et al., 2005; Rodda et al., 2005), a pluripotent cell-specific Sox element binding protein (PSBP) (Kuroda et al. 2005), Tcf3 (Pereira et al.,
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2006), and p53 (Lin et al., 2005). The overexpression of Nanog, therefore, can occur due to more efficient interactions of positive regulators such as Oct3/4-Sox2 (Fig. 4A, panel I) at the nanog promoter. Alternatively, retinol may induce the expression of yet unknown transcription factor/s that interact with nanog promoter either alone (Fig. 4A, panel II) or with Oct3/4-Sox2. To characterize such interactions, (siRNA) for oct3/4 gene were introduced into ESCs using lipofectamine. In spite of over 50% decrease in oct3/4 message, there was no change in the nanog mRNA (Fig. 4B, middle lanes). Whereas, the addition of retinol caused over twofold increase in the nanog message (right lanes) indicating that Nanog overexpression is independent of Oct3/4. The data was further confirmed by Western blot analysis that showed that in spite of approx. 60% decrease in Oct3/4 protein, no effect was observed on Nanog overexpression (Fig. 4C, right lanes). Time course of Nanog activation
Fig. 3 Retinol induced the expression of pluripotent cell-specific genes. R1 embryonic stem cells (ESCs) cultured over mouse embryo fibroblast feeders were treated with different concentrations of retinol using embryonic stem medium. After 24 hr, the ESCs were harvested by trypsinization followed by the separation of feeder cells. (A) Total RNA was analyzed for different genes by reverse transcriptase-polymerase chain reaction using primers specific for the undifferentiated cells and using primers for hypoxanthine guanine phosphorybosyl transferase (hprt) as control. The density of the amplified fragments was quantitated by Bio-Rad Fluor-S-MultiImager and Quantity one-4.2.3 software. (B) Total proteins were analyzed by Western blot analysis using antibodies specific for Nanog and Sox2. (C) Western blot analysis of total proteins using antibodies specific for Oct3/4 and Stat3 using b-Actin as control. The proteins were visualized by Chemi-luminescence and quantitated using Bio-Rad Fluor-S-MultiImager and Quantity one-4.2.3 software. Retinol caused the concentration-dependent increase in only Nanog.
In order to study the earliest effect of retinol on Nanog synthesis, total RNA from ESCs cultured with 0.25 mM retinol for 15, 30, 60, 90, and 120 min was analyzed by RT-PCR analysis. There was a twofold increase in nanog message within the first 60 min of the treatment that increased only slightly after 2 hr, whereas no change was observed in the oct3/4 or stat3 message (Fig. 5A). The Western blot analysis of the same samples revealed a twofold increase in Nanog within first 60 min of the treatment (Fig. 5B). The level peaked to about threefolds in 4 hr and remained steady thereafter. This leads to the conclusion that retinal-induced signaling is highly efficient in up-regulating the nanog expression and the activation of Nanog occurs shortly after the treatment. To prove the expression of Nanog at cellular level, the retinol-treated cells were immunostained using Nanog-specific antibodies. Figure 5C shows that 5-day retinol treatment caused a dramatic increase in Nanog expression at 0.25 and 0.5 mM concentration (panels IV and VI, respectively) as compared with untreated cells (panel II). Enhanced-expression of Nanog is not mediated through retinoic acid Normally, the retinol is oxidized to retinaldehyde in cells by alcohol dehydrogenase 4 (ADH4), which is then oxidized to RA by retinaldehyde dehydrogenase (RALDH-2) (Zhao et al., 1996). ADH4, however, is not detectable until E6.5 during development (Ang and Duester, 1997). RA receptors (retinoic acid receptors [RARs] and retinoid X receptors [RXRs]) are expressed in ESCs and throughout the developing embryo (Hofmann and Eichek, 1994; Chen and Gudas, 1996; Ross et al., 2000). To investigate whether the effect of retinol
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Fig. 4 Overexpression of Nanog by retinol is independent of Oct3/ 4. (A) As nanog promoter contains Oct3/4 and Sox2 binding sites, retinol may induce certain factors that may cause more efficient binding of Sox2-Oct3/4 to nanog promoter (I) or the retinol-induced factors may bind directly to the nanog promoter (II). (B) oct3/4 siRNAs were introduced into ESCs. Reverse transcriptasepolymerase chain reaction analysis of RNA from cells treated with
retinol for 10 hr (Si1Re) and untreated cells (Si). Normal-untreated cells were used as control. (C) Western blot analysis on total proteins isolated from the same cells, treated with retinol for 10 hr (Si1Re) and untreated cells (Si) using antibodies specific for Oct3/4 and Nanog. The overexpression of Nanog protein and mRNA is obvious in spite of decrease in OC3/4. Non-specific siRNAs were used as control for both the experiments.
on Nanog occurs via RA-mediated signaling mechanism; ESCs were treated with 0.25 mM retinol and 1.0 mM RA. After 5 days the cells were tested for alkaline phosphate staining. As shown in Figure 6 (panel III), the retinoic acid caused complete differentiation of ES cells within 5 days. On the other hand, 100% of the colonies remained undifferentiated after treatment with retinol (panel II), whereas over 70% of the cells differentiated when the retinol was supplemented with 1 mM retinoic acid (panel IV) providing indirect evidence that retinol is not metabolized to RA. This also proves that RA and retinol follow independent pathways in ESCs. Furthermore, it is also clear from the data that retinol is unable to counter the RA-mediated differentiation.
were then transferred to the pseudopregnant mothers and the embryos were recovered at different stages such as E7.5 and E16.0 (mid-gestation period). The embryos were then visualized under UV light for ESC integration. Retinol-treated ESCs retained complete potential to contribute to the embryo and extra-embryonic membranes (Fig. 7A, top right panel) and showed high penetration in all the organs (bottom right panel). Further, the cells were also capable of generating live chimeras (Fig. 7B), confirming that retinol does not impair ESC potential for contributing to the different tissues of the embryo. Similarly KBL2 ESCs derived from C57BL6 embryo also produced highly chimeric animals when injected into FVB/N (albino) embryos after treatment with retinol (Fig. 7C).
Retinol does not impair embryo colonization properties of ESCs Pluripotent ESCs have the complete potential to colonize tissues derived from all the primary germ layers. To investigate whether retinol affects their ability to contribute to the various tissues, R1 ESCs were prepared that contained a gene for enhanced green florescence protein (EGFP) stably integrated into their genome (Khillan J. S., unpublished results). The cells were propagated for five passages in the medium supplemented with 0.25 mM retinol followed by aggregation with 2.5 days morulas (Khillan and Bao, 1997). The blastocysts developed from the aggregated embryos
Discussion To sustain their self-renewal, ESCs require a circuitry of different signaling pathways including LIF/Stat3 pathway, BMPs mediated activation of Ids, Wnt/b-catenin signaling, POU-domain-containing oct3/4, and homeodomain-containing sox2 and nanog genes (Smith, 2001). Both Oct3/4 and Nanog are expressed in the early stages in the ICM and the epiblast of the developing embryo. Gene knock-out studies in mouse have demonstrated that Oct3/4 and Nanog are essential for selfrenewal of ESCs (Nichols et al., 1998; Mitsui et al.,
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Fig. 5 Rapid induction of Nanog expression by retinol. Embroynic stem cells cultured over the feeder cells were treated with 0.25 mM retinol for 15min–6 hr. At each interval the cells were treated with lysis buffer. (A) Total RNA isolated from the cells was amplified by reverse transcriptase-polymerase chain reaction. A significant increase in nanog message was observed in 60 min of treatment. (B) Western blot analysis on total proteins from the cells treated with 0.25 mM retinol for 30 min to 6 hr. At each specific time point, the cells were analyzed using Nanog-specific antibodies. Approximately two-fold increase in Nanog was seen at protein level after 1 hr treatment with retinol. (C) Immunostaining of retinol-treated cells. Embryonic stem cells were cultured with 0.25 and 0.5 mM retinol for 5 days. The cells were then immunostained using antibodies against Nanog. A strong Nanog expression was observed in the cells treated with retinol (panels IV and VI) compared with the untreated cells (panel II).
2003). In the developmental hierarchy, nanog is expressed after oct3/4 (Pesce and Scholer, 2001). The first sign of nanog message is seen in the compacted morulae. Inactivation of oct3/4 results in the differentiation of ICM into trophectoderm (Nichols et al., 1998) whereas
nanog inactivation results in the ICM that fails to form epiblast and produces only parietal endoderm-like cells (Mitsui et al., 2003). Although the nanog expression is unaffected by the absence of oct3/4, a sustained expression of oct3/4 is essential for self-renewal of ESCs. The addition of LIF, however, can have an enhancing effect on their growth (Chambers et al., 2003). Recently, Chambers et al. (2003) and Darr et al. (2006) have shown that overexpression of Nanog can promote the self-renewal of ESCs thus indicating that it is important for maintaining the pluripotency. However, no effect on the cell proliferation was reported. Our studies have shown that addition of retinol to the culture medium did not increase the proliferation of ESCs as the cell number between treated and untreated cultures remained almost same in spite of threefold overexpression of Nanog even after six passages (data not shown). Recently, Zhang et al. (2005) reported that expression of Nanog promotes the proliferation of NIH 3T3 cells. Using three independent cell lines such as Cos 7, HEK293, and NIH3T3, Peistun et al. (2006), on the other hand, reported that Nanog can transform NIH3T3 cells. They further showed that Nanog targets specific set of genes that is unique to each cell type. In this respect, the overexpression of Nanog in ESCs may only be responsible for maintaining the optimal level of pluripotency-specific genes. This phenomenon needs further investigation. Overexpression of Nanog without any effect on Stat3 supports the earlier observations that Nanog can bypass the LIF/Stat3 pathway, though a certain level of Nanog is critical for maintaining the cytokine-independent selfrenewal (Chambers et al., 2003). A threefold increase in Nanog by retinol, therefore, appears to satisfy that requirement. However, further studies are needed to prove this. Under normal conditions, the ESC cultures require trypsinization every 3–4 days to break up the colonies to prevent differentiation. Retinol, on the other hand, supported the growth of undifferentiated cells for 12 days. The effect of retinol beyond 12 days was not tested. Further analysis of cells by RT-PCR for differentiation-specific genes fgf8, nestin, and brachyury did not show amplification of the specific fragments suggesting the undifferentiated nature of the cells. Although the ESCs exhibited a dose-dependent response, the net effect of retinol remained the activation of Nanog. In all the cultures described here, the retinol concentrations used were less than the physiological levels (1.0–2.0 mM) (Lane et al., 1999). A retinol concentration as high as 2.0 mM also did not show any obvious toxic effect on ESCs; however, the cells started to bleb after one to two passages. Therefore, for all the subsequent analyses we used only 0.25 mM concentration, which was sufficient to maintain the pluripotency of ESCs. Nanog function is conserved between species; for example human NANOG can direct LIF independent
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Fig. 6 Suppression of Embryonic stem cells (ESC) differentiation by retinol does not involve signaling by retinoic acid. ESCs cultured over mouse embryo fibroblast feeder cells were treated with 0.25 mM retinol in the absence (Re) and presence of 1.0 mM retinoic acid (RA) (Re1RA). Fresh medium was used everyday with
fresh retinol and retinoic acid. The cells were stained for alkaline phosphatase after 5 days. Addition of retinoic acid caused differentiation of ESCs in the presence (panel IV) or absence (panel III) of retinol. Almost all the cells remained undifferentiated in retinolcontaining medium (panel II). Control cells (panel I).
self-renewal of mouse and monkey ESCs (Chambers et al., 2003; Yasuda et al., 2006). Mouse Nanog has 87% and 58% identity with rat and human orthologs, respectively. This sequence identity is more pronounced in the homeodomain region (94% for rat and 87% for humans) (Chambers et al., 2003). Nanog may sustain the pluripotency of ESCs either by activating the undifferentiated cell-specific genes and/or by the suppression of differentiation-promoting genes chromatin immuno precipitation (ChIP)- pair end ditag (PET) assay revealed many genes that contain Nanog binding sites (Loh et al., 2006). Differentiation-specific genes such as gata4 and gata6 contain DNA recognition motif of Nanog and their expression is up-regulated in Nanog null cells (Mitsui et al., 2003). Our data clearly demonstrate that the retinol-mediated activation of nanog is independent of known signaling pathways including Jak/Stat3 pathway, BMP/Id, Wnt/b-catenin signaling, and Oct3/4-Sox2 suggesting the possibility of an alternate signaling mechanism by retinol. Though the binding of Oct3/4-Sox2 at the conserved binding sites in the nanog promoter have been shown to regulate its expression (Kuroda et al., 2005; Rodda et al., 2005), our RNAi data suggest that retinol effect may not occur via this mechanism and may involve some unknown transcription factors. Further, in spite of increases in their message, the Oct3/4 and Sox2 levels remained unchanged suggesting a control at the translation level. In this respect it is worth noting the studies described by Kuroda et al. (2005) where they demonstrated that the expression of a reporter gene from nanog promoter is 15% higher when 2.5 kb upstream region was used as compared with Oct3/4Sox2 binding sites containing 353 bp region indicat-
ing the involvement of other factors in nanog regulation. Our studies, however, do not completely rule out the role of Oct3/4-Sox2 as after repeated attempts we were unable to eliminate the oct3/4 message. On the other hand, the absence of any change in fgf4 levels, a gene that also contains binding sites for Oct3/4 and Sox 2 (Kuroda et al., 2005), lends support for Oct3/4- and Sox2-independent mechanism. Retinol-mediated overexpression may occur either due to the interaction of some unknown transcription factor/s with Oct3/4-Sox2 (Fig. 4, panel I) or alternatively via binding of this factor/s to some specific regulatory elements in the nanog promoter (panel II). The studies are currently in progress to identify such a potential element/s in the nanog promoter. However, with the available data we cannot completely rule out the role of these two factors. Further, we have not yet tested the auto-regulation by Nanog for which we are generating gene constructs with different promoter lengths and reporter luciferase gene. Retinol, the alcohol form of vitamin A, and its derivatives play an essential role in many biological functions during development of the embryo. Thus far no specific biological role has been assigned to retinol as such. With the exception of retinaldehyde, which is involved in visual cycle (Wald, 1968), the biologically active form of retinol is RA that regulates many differentiation specific genes by binding to the RAR and RXR (Balmer and Blomhoff, 2002; Clagett-Dame and DeLuca, 2002). A notable example of ESC differentiation by retinol metabolites was reported by Lane et al., (1999). A careful review of these studies revealed a critical difference in the methodology to culture the cells that affected the outcome of their investigations. These
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Fig. 7 Retinol does not affect the integration potential of embryonic stem cells (ESCs) into different tissues. Green florescence protein (EGFP)-positive R1 ESCs and C57BL6 ESCs were cultured in the presence of 0.25 mM retinol for five passages. (A) C57BL6 embryos were microinjected with R1ESCs and the embryos were recovered from mothers at E7.5 and E16.0. The embryos and fetuses
were visualized under UV light. An arrow in the top left panel shows the direction of the uterine attachment. (B) Chimeric animal generated by aggregation of C57BL6 morula and R1ESCs. (C) Chimeric pups generated by microinjection of C57BL6 ESCs into FVB/N (albino) blastocysts.
investigators cultured ESCs without feeders and LIF for 96 hr before the addition of retinol. Under such conditions, the ESCs are expected to be already in the state of differentiation. In the current studies, the retinol was added almost immediately after the cells had settled down. Our data also rule out the possibility of involvement of RA that suggests a provocative hypothesis that the suppression of differentiation in ESCs may be caused by some novel signaling mechanism mediated via yet unknown retinol metabolite/s. Analysis of the retinoltreated ESCs by HPLC analysis may reveal the identity of such molecules. The alternate pathway used by retinol to suppress differentiation may be important to maintain the functional balance of retinol during embryonic and fetal development. The regulation of nanog by a physiologically relevant compound such as retinol offers a unique opportunity
to identify transcription factors, which control its expression that may lead to much needed information about the master regulatory genes responsible for the ‘‘stemness’’ of the cell. Colonization of retinol-treated ESCs into embryos, extra-embryonic membranes, and chimeric animal proves that retinol does not affect the pluripotency of ESCs. Until now, very little information is available on the in vivo and in vitro role of retinol on embryo development. Recently, Livingston et al. (2004) demonstrated that retinol can improve the development of bovine embryos in vitro. Studies are currently in progress in our lab to investigate the in vitro effect of retinol on preimplantation embryo as well as post-implantation embryo in vivo after transfer of retinol-treated embryos into pseudopregnant mothers. Overexpression of Nanog by retinol may be of great significance for the development
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and stabilization of new ES cell lines from human embryos especially from the embryos generated for therapeutic cloning after somatic cell nuclear transfer. Studies are in progress to investigate whether the human NANOG also responds to retinol in a similar fashion. Our preliminary studies have shown that human ESCs form compact colonies following retinol treatment.
Acknowledgments We thank Dr. Arthur S. Levine, Dean of Graduate Studies, University of Pittsburgh, for providing the support for these studies and Drs. Devjani Chatterjee and Sanjay Mishra for critical reading of the manuscript. We also thank Dr. P. Monga for providing antibodies for b-catenin.
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