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First published online July 19, 2007
Stem Cells Vol. 25 No. 11 November 2007, pp. 2705 -2711
doi:10.1634/stemcells.2007-0086; www.StemCells.com
© 2007 AlphaMed Press

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EMBRYONIC STEM CELLS

Efficient Derivation of Embryonic Stem Cells by Inhibition of Glycogen Synthase Kinase-3

Hiroki Umeharaa, Tohru Kimurab, Satoshi Ohtsukac, Toshinobu Nakamuraa, Kenji Kitajimab, Masahito Ikawad, Masaru Okabed, Hitoshi Niwac,e, Toru Nakanoa,b

aGraduate School of Frontier Biosciences,
bDepartment of Pathology, Graduate School of Medicine, Osaka University, Osaka, Japan;
cLaboratory for Pluripotent Cell Studies, RIKEN Center for Developmental Biology, Kobe, Japan;
dGenome Information Research Center, Institute for Microbial Diseases, Osaka University, Osaka, Japan; and
eDepartment of Developmental and Regenerative Medicine, Graduate School of Medicine, Kobe University, Kobe, Japan

Key Words. Embryonic stem cells • Glycogen synthase kinase-3 • Akt • Pluripotency • Stem cells

Correspondence: Toru Nakano, M.D., Ph.D., Department of Pathology, Graduate School of Medicine, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka, Japan 565-0871. Telephone: 81-6-6879-3720; Fax: 81-6-6879-3729; e-mail: tnakano{at}patho.med.osaka-u.ac.jp

Received on January 31, 2007; accepted for publication on July 9, 2007.

First published online in STEM CELLS EXPRESS  July 19, 2007.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem (ES) cells are derived from the inner cell mass (ICM) of blastocysts. The use of ES cells as a source of differentiated cells holds great promise for cell transplantation therapy. The efficiency of ES cell derivation is affected by genetic variation in mice; that is, some mouse strains, such as C57BL/6, are amenable to ES cell derivation, whereas others, such as BALB/c, are refractory. Developing an efficient method to establish ES cells from strains of various genetic backgrounds should be valuable for derivation of ES cells in various mammalian species, including human. Although it is well-established that various signaling pathways, including phosphoinositide 3-kinase (PI3K)/Akt and Wnt/β-catenin, regulate the maintenance of ES cell pluripotency, little is known about the signaling pathways involved in the derivation of ES cells from ICMs. In this study, we demonstrated that inhibition of glycogen synthase kinase-3 (GSK-3), one of the crucial molecules in the regulation of the Wnt/β-catenin, Hedgehog, and Notch signaling pathways, dramatically augmented ES cell derivation from both C57BL/6 and BALB/c mouse strains. In contrast, Akt signaling activation enhanced the growth of ICM but did not increase the efficiency of ES cell derivation. Our study establishes an efficient means for ES cell derivation by pharmacological inhibition of GSK-3.

Disclosure of potential conflicts of interest is found at the end of this article.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Embryonic stem (ES) cell lines have been established from several mammalian species, including mouse, monkey, and human [1, 2]. Although ES cell lines have been derived in many laboratories, the success rate for derivation is variable, probably reflecting differences in embryo quality, derivation methods, and genetic background [3]. For instance, mouse strains such as C57BL/6 are amenable to establishing ES cell lines, whereas strains such as BALB/c, Institute for Cancer Research, and nonobese diabetic are refractory [4, 5].

The mechanisms that sustain ES cell pluripotency in culture have been investigated extensively. Pluripotency can be maintained by leukemia inhibitory factor (LIF) in mouse ES cells. The downstream activation of signal transducer and activator of transcription-3 (STAT3) is essential for LIF action [6–8]. Despite its crucial roles in mice, STAT3 activation is not involved in the maintenance of primate ES cell pluripotency [9, 10]. In contrast, the pluripotency of both mouse and human ES cells can be maintained by inhibition of glycogen synthase kinase-3 (GSK-3), which was revealed by its specific inhibitor [11]. Activation of Wnt/β-catenin signaling is caused by the GSK-3 inhibitor and could play important roles in the maintenance of ES cell pluripotency. However, the involvement of Wnt/β-catenin signaling in the maintenance of ES cell pluripotency is controversial, because addition of recombinant Wnt3a stimulates human ES cell differentiation and is not sufficient for maintaining mouse ES cell pluripotency [12, 13]. Other GSK-3 targets, such as the Hedgehog and Notch signaling pathways, may cooperatively regulate pluripotency. Meanwhile, the activation of phosphoinositide 3-kinase (PI3K) and the downstream serine-threonine kinase Akt plays a critical role in the maintenance of ES cell pluripotency. Pharmacological inhibition of PI3K induces differentiation of mouse ES cells, and introduction of the active form of Akt maintains pluripotency in mouse and primate ES cells [14, 15].

In contrast to the extensive study on the molecular basis for sustaining ES cell pluripotency [16, 17], the mechanisms underlying the derivation of ES cell lines from epiblasts remain poorly understood. In this study, we investigated the effects of GSK-3 and Akt signaling on the derivation of ES cells from the inner cell mass (ICM). Inhibition of GSK-3 signaling but not activation of Akt signaling augmented the efficiency of ES cell derivation in mice. Our pharmacological approach using a GSK-3 inhibitor potentially provides a general strategy to improve ES cell derivation from diverse mammalian species.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Mice and Embryos
Two-cell embryos were obtained from C57BL/6 and BALB/c inbred strains. Embryos were also isolated from a cross between Akt-Mer transgenic male mice of a C57BL/6-DBA2 mixed background and C57BL/6 female mice [18]. Female mice were superovulated by injection with pregnant mare serum gonadotropin (ASKA Pharmaceutical, Tokyo, http://www.aska-pharma.co.jp) followed by injection with human chorionic gonadotropin (ASKA Pharmaceutical) 48 hours later. Mice were kept on a 12-hour-light/12-hour-dark regimen. Twelve hours from the middle of the dark period was termed embryonic day 0.5 (E0.5). E13.5 fetuses from the ICR strain were used to obtain mouse embryonic fibroblasts (MEFs) for use as feeder cells. All animal studies were approved by the Animal Care and Use Committee of Osaka University.

Isolation of ICM
Two-cell-stage embryos (E1.5) were flushed from the oviducts into phosphate-buffered saline (PBS) with 10% fetal calf serum (FCS) and cultured for 3 days in Hepes-buffered potassium simplex-optimized medium (KSOM; Specialty Media, Phillipsburg, NJ, http://www.specialtymedia.com/) or KSOM supplemented with 4-hyroxytamoxifen (4OHT; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Immunosurgery was performed on blastocysts as described previously [19]. Briefly, the zona pellucida was removed with acid-Tyrode's solution (Sigma-Aldrich), and the blastocysts were treated with rabbit anti-mouse red blood cell antibody (Inter-cell Technologies, Jupiter, FL) for 30 minutes in a CO2 incubator at 37°C. After three washes with KSOM, embryos were treated with guinea pig complement (Merck, Darmstadt, Germany, http://www.merck.com) diluted in KSOM. Embryos were observed periodically under a dissecting microscope to check for lysis of trophectodermal cells. We usually observed membrane blebbing within 30 minutes. The ICM was then isolated and used for derivation of ES cell lines.

Derivation and Culture of ES Cell Lines
The ES culture medium was Glasgow minimal essential medium supplemented with 20% knockout serum replacement (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com), 0.3% FCS (Sigma-Aldrich), 2-mercaptoethanol (Sigma-Aldrich), antibiotics (Sigma-Aldrich), nonessential amino acids (Gibco-BRL), pyruvate (Gibco-BRL), and LIF. 6-Bromoindirubin-3'-oxime (BIO; Merck), methyl-BIO (Me-BIO; Merck), or 4OHT was supplemented in the ES medium. The isolated ICMs were seeded onto MEFs and cultured for 6 days. The expanded ICMs were dissociated individually into fresh wells and cultured for 5 days. When ES colonies were visible, the cells were trypsinized and replated in 35-mm dishes with fresh feeders.

Immunostaining
Cells were fixed with 4% paraformaldehyde (PFA) in PBS for 10 minutes at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 10 minutes. The blocking solution consisted of 5% normal goat serum with 0.2% Tween 20 in PBS and was used for 30 minutes at room temperature. Anti-Oct-3/4 (1:200; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), stage-specific embryonic antigen-1 (1:50; Kyowa Hakko, Tokyo, http://www.kyowa.co.jp/eng/), {alpha}-smooth muscle actin (DAKO, Glostrup, Denmark, http://www.dako.com), {alpha}-fetoprotein (DAKO), type II collagen (1:100; Chemicon, Temecula, CA, http://www.chemicon.com), and pancytokeratin (1:200; Sigma-Aldrich) were used as primary antibodies. Alexa Fluor 568-conjugated and Alexa Fluor 488-conjugated antibodies (1:200; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) were used as secondary antibodies to detect primary antibody complexes.

In Vitro Differentiation Assays
In vitro differentiation induction of ES cells into hematopoietic cells was performed as previously described [20]. For EB formation, ES cells were trypsinized, resuspended in ES culture medium without LIF, and replated on bacteriological dishes at a density of 1.6 x 105 cells per cm2. Cells were refed every second day and cultured for 7 days in suspension. The resultant cystic EBs were then adhered to gelatin-coated dishes and cultured for an additional 7 days. Total RNA was prepared from the EBs at days 0, 3, 5, 7, 10, and 14 after differentiation induction.

Reverse Transcription-Polymerase Chain Reaction
Total RNA was isolated by an RNeasy mini kit (Qiagen, Valencia, CA, http://www1.qiagen.com), and 1 µg of total RNA was used for cDNA synthesis. The reverse transcription was performed using the ThermoScript reverse transcription-polymerase chain reaction (RT-PCR) system (Gibco-BRL) as described [21]. PCRs were optimized to allow semiquantitative comparisons within the log phase of amplification. The primer sequences and amplification cycles are listed in Table 1. The PCR products were analyzed by agarose gel electrophoresis and visualized using ethidium bromide staining. The intensity of bands was quantified by NIH ImageJ (http://rsb.info.nih.gov/nih-image) and normalized against GAPDH.


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  		Table 1. Oligonucleotides primers used for reverse transcription-PCR analysis

 
Teratoma Formation
ES cells (5 x 106) were injected subcutaneously into nude mice. After 3 weeks, the teratoma were excised, fixed in 4% PFA, and subjected to histological examination with H&E, periodic acid-Schiff (PAS), Alcian Blue, and immunostaining.

Production of Chimeric Mice
ES cells derived from C57BL/6 (coat color, black) and BALB/c (coat color, white) strains were injected into the blastocele of recipient blastocyst-stage embryos from BALB/c and C57BL/6 strains, respectively. The incorporation of cells into the chimeric mice was monitored by coat color. Germline transmission was then tested by crossing the chimeras with ICR strains (coat-color genotype, AA/bb/cc). Germline transmission of the C57BL/6 cells (aa/BB/CC) produces the offspring with agouti coat color (Aa/Bb/Cc), whereas the transmission of the BALB/c cells (AA/bb/cc) produces offspring with white coat color (AA/bb/cc).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 
Augmentation of ES Cell Derivation by Pharmacological Inhibition of GSK-3
We investigated the impact of GSK-3 inhibition on ES cell derivation using the specific inhibitor BIO or its inactivated derivative, Me-BIO [22]. The experimental scheme for ES cell derivation used in this study is illustrated in Figure 1A. Two-cell-stage embryos were recovered at E1.5 from superovulated C57BL/6 female mice and cultured for 3 days to the blastocyst stage without BIO. The ICMs were isolated by immunosurgery and plated onto feeder layers of MEFs. Culture of the ICMs was conducted in the presence of LIF and BIO until the establishment of ES cell lines. At day 6 after seeding on MEFs, approximately 30% of the ICMs attached and proliferated to form large expanded ICM colonies, which consisted of proliferating epiblasts inside and growing primitive endoderm outside (Fig. 1B; Table 2). In the culture treated with 2 µM BIO, approximately 80% of the ICMs expanded to form colonies, and outgrowth of the primitive endoderm was suppressed (Fig. 1B). The expanded ICMs were picked up, trypsinized, and passed onto MEFs to establish ES cell lines. When treated with BIO, nearly all of the expanded ICM colonies (34 of 35) gave rise to ES cell lines (Fig. 1B; Table 2). In total, the efficiency of ES cell derivation increased by approximately fourfold with 2 µM BIO treatment in C57BL/6 mice.


Figure 1
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  		Figure 1. ES cell derivation by treatment with glycogen synthase kinase-3 inhibitor. (A): Experimental scheme of ES cell derivation. Two-cell-stage embryos were developed to the blastocyst stage by culturing for 3 days in potassium simplex-optimized medium in vitro. The ICMs isolated from blastocysts by immunosurgery were seeded onto mouse embryonic fibroblasts (MEFs) and allowed to expand for 6 days. The expanded ICMs were dissociated individually onto fresh MEFs and cultured for 5 days. The colonies with ES-like morphology were then selected for further propagation and characterization. BIO treatment was started at the beginning of the ICM culture and was continued until ES establishment. (B): Expanded ICMs (left and middle) and ES cell colonies (right) derived from C57BL/6 mice cultured in the presence of 2 µM Me-BIO and 2 µM BIO. BIO treatment did not affect proliferation of epiblasts but suppressed endodermal growth in the ICM cultures. Arrow and arrowhead represent the epiblasts and endodermal cells, respectively. (C): Expanded ICM colonies derived from BALB/c mice cultured in the presence of 2 µM Me-BIO, 2 µM BIO, and 5 µM BIO. Augmentation of ES cell derivation was correlated with the suppression of endodermal cells. Arrow and arrowhead represent the epiblasts and endodermal cells, respectively. (D): ES cell lines established from BALB/c mice in the presence of 5 µM BIO. These cells expressed undifferentiated-ES-cell markers, such as ALP, Oct-3/4, and stage-specific embryonic antigen-1. Scale bars = 100 µm (B–D). Abbreviations: ALP, alkaline phosphatase; BIO, 6-bromoindirubin-3'-oxime; ES, embryonic stem; ICM, inner cell mass; Me-BIO, methyl-6-bromoindirubin-3'-oxime.

 


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  		Table 2. Efficiency of ES cell derivation by treatment with glycogen synthase kinase-3 inhibitor

 
The BALB/c mouse strain is reportedly refractory for ES cell derivation [4, 5]. Indeed, ES cell colonies emerged from the ICM culture of BALB/c embryos, but only a small number of ES cell lines could be stably propagated (Table 2). Similarly, treatment with 2 µM BIO did not facilitate ES cell derivation in BALB/c embryos. The endodermal cells in the BIO-treated ICM colonies proliferated extensively, as did control ICMs (Fig. 1C). However, when the concentration of BIO was increased to 5 µM, endodermal outgrowth was suppressed (Fig. 1C) and ES cell lines were established from more than 30% of the embryos (Table 2). Once established, these cells proliferated indefinitely, retaining the characteristics of undifferentiated ES cells in the absence of BIO (Fig. 1D). These results clearly demonstrate that ES cell derivation is augmented by inhibition of GSK-3, regardless of genetic background.

We next examined the effects of Akt signaling activation on the efficiency of ES cell derivation (details given in supplemental online data). For this purpose, we used transgenic mice expressing the Akt-Mer chimeric protein (C57BL/6-DBA2 mixed background) [18], which allowed us to control Akt signaling activation in a conditional manner by the addition or removal of 4OHT. When Akt signaling was activated from the two-cell stage, both the cell size and the cell number increased in the isolated ICMs (supplemental online Fig. 1). However, the efficiency of ES cell derivation was not altered by the activation of Akt even in the case in which activation was induced during the entire derivation process (supplemental online Table 1).

Multipotent Differentiation Capacities of the Established ES Cell Lines
Differentiation capacities of the ES cell lines established with BIO were first evaluated by in vitro differentiation assays. The EBs were generated from the ES cells established from both C57BL/6 and BALB/c strains. As shown in Figure 2A, the vast majority of EBs did not contain the Oct3/4-positive cells at day 6 after differentiation induction, indicating that differentiation took place uniformly in the EBs. There were no significant differences between the ES cells that were established in the presence of BIO and those established in the presence of Me-BIO. {alpha}-Fetoprotein (AFP)-positive cells and {alpha}-smooth muscle actin-positive cells were detected in some fraction of EBs, as shown in Figure 2B.


Figure 2
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  		Figure 2. Differentiation of ES cells established with BIO in EB formation assay. (A): Differentiation status of the EBs generated from the ES cells established with Me-BIO or BIO. The EBs at day 6 after differentiation induction were stained with anti-Oct3/4 antibody. The EBs that had a small number of Oct3/4-positive cells were counted as partially differentiated EBs. Oct-3/4-positive cells were not detected in large portions of the EBs. The ES cells cultured with leukemia inhibitory factor were stained as a positive control. (B): Double immunostaining of the day 6 EBs with the antibodies against differentiation markers (AFP and {alpha}-SMA; red) and anti-Oct3/4 antibody (green). The EBs generated from C57BL/6 (left column) and BALB/c (right column) mouse strains were positive for AFP (top) and SMA (bottom). The signals for Oct-3/4 were not detectable. Scale bar = 100 µm. Abbreviations: AFP, {alpha}-fetoprotein; BIO, 6-bromoindirubin-3'-oxime; ES, embryonic stem; Me-BIO, methyl-6-bromoindirubin-3'-oxime; SMA, {alpha}-smooth muscle actin.

 
To determine the differentiation capacities quantitatively, we examined the marker gene expression in a long-term EB differentiation system. The ES cells were applied to suspension cultures to produce EBs for 7 days and then adhered to gelatin-coated plates and differentiated for an additional 7 days. Expression of the marker genes for three germ layers was analyzed by semiquantitative RT-PCR. We examined Mixl1 and Brachyury as mesodermal markers, Collagen IV and AFP as endodermal markers, and Nestin as an ectodermal marker. As shown in Figure 3, the time courses and the maximal levels of the expression were slightly varied among the cell lines; expression of all the differentiation markers was efficiently induced in the ES cell lines established with BIO. Furthermore, in an in vitro hematopoietic differentiation system, the ES cells also differentiated into mesodermal colonies and then to various hematopoietic cells (Fig. 4A).


Figure 3
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  		Figure 3. Expression of differentiation marker genes in the EB formation assay. Semiquantitative RT-polymerase chain reaction (RT-PCR) analysis of EBs generated from the embryonic stem (ES) cell lines established from C57BL/6 (A) and BALB/c (B) strains. Expression of the marker genes of three germ layers was analyzed by RT-PCR. The PCR products were run on agarose gel and visualized by staining with ethidium bromide. The intensity of each band was quantified and normalized against GAPDH expression. Mixl1 and Brachyury, mesoderm markers; Collagen IV and AFP, endoderm markers; Nestin, ectoderm marker. Abbreviations: AFP, {alpha}-fetoprotein; BIO, 6-bromoindirubin-3'-oxime; Me-BIO, methyl-6-bromoindirubin-3'-oxime; RT, reverse transcription.

 


Figure 4
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  		Figure 4. Multipotent differentiation capacity of embryonic stem (ES) cells established in the presence of 6-bromoindirubin-3'-oxime. (A): Hematopoietic differentiation capacities. The established ES cell lines were applied to an in vitro hematopoietic cell differentiation system using OP9 stromal cells. ES cells derived from both the C57BL/6 (left column) and BALB/c (right column) mice generated mesodermal colonies by day 5 (top) and differentiated to a variety of hematopoietic cells by day 12 (bottom) after differentiation induction. Scale bar = 40 µm. (B–D): Teratomas generated from the established ES cell lines. The ES cell lines from C57BL/6 (left column) and BALB/c (right column) mouse strains were injected under the skin of nude mice. After 3 weeks, the teratomas contained various tissues, including cartilage (B) (H&E, Alcian Blue, and anti-type II collagen staining), mucosal glands (C) (H&E, periodic acid-Schiff, Alcian Blue, and anti-cytokeratin staining), and squamous epithelium (D) (H&E and anti-cytokeratin staining). Scale bars = 100 µm. (E): Production of chimeric mice using the established ES cells. ES cells derived from C57BL/6 mice (black coat) were injected into blastocysts of BALB/c mice (white coat; left) or ES cells derived from BALB/c mice were injected into blastocysts of C57BL/6 mice (right). (F): Germline transmission of a C57BL/6 ES cell line. Chimeras obtained after injection of the C57BL/6 ES cells into BALB/c blastocysts were crossed to ICR mice. Germline transmission of the C57BL/6 cells was demonstrated by the birth of offspring with agouti coat color (arrowhead; details are given in Materials and Methods).

 
We next analyzed the multilineage differentiation capacities of the ES cell lines in vivo. When transplanted into nude mice, the ES cells established from both the C57BL/6 and the BALB/c strains produced various differentiated tissues, such as cartilage, mucosal gland, and epithelium (Fig. 4B–4D). The cartilage cells expressed chondrocyte marker type II collagen, and proteoglycan synthesis was shown by staining with Alcian Blue (Fig. 4B). The cells of mucosal glands expressed the epithelial marker cytokeratin (Fig. 4C). The cavities of the glands contained the materials stained with PAS and Alcian Blue, showing that the gland cells secreted acid mucin. The squamous epithelial cells were positive for cytokeratin and contained terminally differentiated stratum corneum (Fig. 4D). Thus, the ES cells established with BIO had the capacity to produce various tissue structures. Next, we examined the capacity to contribute to chimeric mice. Five C57BL/6 ES cell lines were injected into E3.5 BALB/c blastocysts. Two cell lines generated chimeric mice (Fig. 4E), and one line was transmitted through germline (Fig. 4F). The chimeric mice were also produced from two BALB/c ES cell lines out of three lines examined (Fig. 4E), but no germline transmission was observed. Taken together with the above in vitro and in vivo differentiation assays, our data indicate that the ES cells established by GSK-3 inhibition possessed pluripotent differentiation ability.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosure of Potential...
 Acknowledgments
 References
 
In this study, we analyzed the effects of GSK-3 inhibition and Akt activation on ES cell derivation from ICM cultures. First, we demonstrated that inhibition of GSK-3 with BIO augmented the efficiency of ES cell derivation, regardless of the mouse strain used. BIO treatment dramatically enhanced the efficiency of ES cell derivation from expanded ICMs in both C57BL/6 and BALB/c strains (Table 1). In addition, the emergence of expanded ICM colonies was also promoted by the BIO treatment in C57BL/6 mice. In contrast to GSK-3 inhibition, activation of Akt signaling did not show a supportive effect on ES cell derivation. However, the activation of Akt signaling increased the size and the number of ICM cells. Since the Akt-Mer transgenic mice had the C57BL/6-DBA/2 mixed background, the possibility could not be excluded that the difference in the efficiency of ES cell derivation between GSK-3 inhibition and Akt activation reflects the differences in the genetic background of the mice used.

Multipotent differentiation capacities of the ES cell lines established with BIO were shown by in vitro differentiation assays, teratoma formation in nude mice, and chimeric mouse production. Although the C57BL/6 ES cell line was successfully transmitted through germline, none of the BALB/c ES cell lines showed germline transmission. To our knowledge, only one report has demonstrated the germline transmission of BALB/c-derived ES cells [23]. The chromosomal instability is likely the major reason why BALB/c-derived ES cells failed to transmit through germline. Indeed, the BALB/c ES cell lines showed a higher percentage of aneuploidy than the C57BL/6 ES cells (data not shown). Thus, additional studies should be necessary to develop not only the methods to derive ES cells but also the procedures to maintain the normal karyotype of ES cells stably.

ICM cells differentiate to epiblasts and primitive endoderm. Epiblasts produce the entire fetus after implantation, whereas primitive endodermal cells contribute to the yolk sac. In the ICM cultures for ES cell derivation, a population of epiblasts within the ICM colonies gives rise to ES cell lines while maintaining the pluripotency. Considering the process of ES cell line establishment, BIO treatment presumably supports the pluripotency of epiblasts during the initial phase of culture.

How does GSK-3 inhibition improve the ES cell derivation? GSK-3 activates various signaling pathways, such as Wnt/β-catenin, Hedgehog, Notch, and protein kinase A (PKA) signals [24, 25]. It has been demonstrated that Wnt/β-catenin signaling is activated in BIO-treated ES cells [11]. However, Wnt/β-catenin signaling alone cannot account for the BIO-mediated maintenance of pluripotency because neither the addition of Wnt3a nor the introduction of stabilized β-catenin was sufficient to maintain the ES cell pluripotency [12, 13]. Hedgehog and Notch signaling pathways are also implicated in self-renewal of tissue stem cells and cancer stem cells [26–30], suggesting that BIO-induced activation of these signals would play important roles in the efficient ES cell derivation.

Besides the signaling pathways, a number of proteins are regulated by GSK-3 through phosphorylation. c-Myc is an attractive candidate that may participate in the promotion of ES cell derivation. Enforced expression of c-Myc supports the mouse ES cell self-renewal in the absence of LIF [31] and reprograms fibroblasts to pluripotent stem cells in cooperation with Oct-3/4, Sox-2, and Klf4 [32]. Because GSK-3 induces c-Myc degradation through its phosphorylation, stabilization of c-Myc induced by GSK-3 inhibition would promote the ES cell derivation. When these data are taken together, it is conceivable that activation of several signaling pathways and stabilization of c-Myc cooperatively support the pluripotency of the epiblasts during the ES cell derivation.

GSK-3 activity is inhibited by Akt via direct phosphorylation. However, Akt activation does not seem to inactivate the entire pool of GSK-3. Introduction of activated Akt into ES cells induces hyperphosphorylation of GSK-3 but does not activate Wnt/β-catenin signaling [15], indicating that Akt activation cannot inactivate a fraction of GSK-3 within the β-catenin destruction complex in ES cells. In addition, GSK-3 is integrated into the protein complexes involved in the Hedgehog and PKA signaling pathways [24, 25]. The insulation of some fraction of GSK-3 from the Akt signal may explain why treatment with GSK-3 inhibitor but not Akt activation augments ES cell derivation.

BIO-augmented ES cell establishment was preceded by the suppression of endodermal outgrowth (Fig. 1B, 1C). The critical role of GSK-3 in primitive endodermal development was also demonstrated by the impaired primitive endodermal growth in blastocysts that had been treated with BIO from the two-cell stage (data not shown). In contrast, Akt activation did not inhibit the outgrowth of endodermal cells in isolated ICM culture (supplemental online Fig. 1). Thus, in addition to its supporting effect on epiblasts, inhibition of GSK-3 with BIO treatment may improve ES cell derivation via inhibition of endodermal proliferation.

In this study, we revealed that ES cells can be established efficiently not only from permissive but also from refractory mouse strains using BIO treatment. Availability of ES cells derived from various mouse strains with genetic disorders and alterations will help us understand the complex genetic networks underlying human diseases and developmental processes. Self-renewal of ES cells is supported by BIO treatment in both mice and humans [11], indicating that GSK-3 plays an evolutionarily conserved role in the regulation of ES cell pluripotency. Hence, this strategy is potentially applicable to ES cell derivation in various mammalian species, including humans.


   DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
References
 
We thank Dr. E. Morii for advices on histological analysis and A. Kawai and Y. Koreeda for production of chimeric mice. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture; Astellas Foundation for Research on Metabolic Disorders; and the 21st Century Center of Excellence, Center for Integrated Cell and Tissue Regulation.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosure of Potential...
 Acknowledgments
 References
 

  1. Prelle K, Zink N, Wolf E. Pluripotent stem cells—Model of embryonic development, tool for gene targeting, and basis of cell therapy. Anat Histol Embryol 2002;31:169–186.[CrossRef][Medline]

  2. Wobus AM, Boheler KR. Embryonic stem cells: Prospects for developmental biology and cell therapy. Physiol Rev 2005;85:635–678.[Abstract/Free Full Text]

  3. Hoffman LM, Carpenter MK. Characterization and culture of human embryonic stem cells. Nat Biotechnol 2005;23:699–708.[CrossRef][Medline]

  4. Kawase E, Suemori H, Takahashi N et al. Strain difference in establishment of mouse embryonic stem (ES) cell lines. Int J Dev Biol 1994;38:385–390.[Medline]

  5. Schoonjans L, Kreemers V, Danloy S et al. Improved generation of germline-competent embryonic stem cell lines from inbred mouse strains. STEM CELLS 2003;21:90–97.[Abstract/Free Full Text]

  6. Boeuf H, Hauss C, Graeve FD et al. Leukemia inhibitory factor-dependent transcriptional activation in embryonic stem cells. J Cell Biol 1997;138:1207–1217.[Abstract/Free Full Text]

  7. Matsuda T, Nakamura T, Nakao K et al. STAT3 activation is sufficient to maintain an undifferentiated state of mouse embryonic stem cells. EMBO J 1999;18:4261–4269.[CrossRef][Medline]

  8. Niwa H, Burdon T, Chambers I et al. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev 1998;12:2048–2060.[Abstract/Free Full Text]

  9. Humphrey RK, Beattie GM, Lopez AD et al. Maintenance of pluripotency in human embryonic stem cells is STAT3 independent. STEM CELLS 2004;22:522–530.[Abstract/Free Full Text]

  10. Sumi T, Fujimoto Y, Nakatsuji N et al. STAT3 is dispensable for maintenance of self-renewal in nonhuman primate embryonic stem cells. STEM CELLS 2004;22:861–872.[Abstract/Free Full Text]

  11. Sato N, Meijer L, Skaltsounis L et al. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004;10:55–63.[CrossRef][Medline]

  12. Dravid G, Ye Z, Hammond H et al. Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. STEM CELLS 2005;23:1489–1501.[Abstract/Free Full Text]

  13. Ogawa K, Nishinakamura R, Iwamatsu Y et al. Synergistic action of Wnt and LIF in maintaining pluripotency of mouse ES cells. Biochem Biophys Res Commun 2006;343:159–166.[CrossRef][Medline]

  14. Paling NR, Wheadon H, Bone HK et al. Regulation of embryonic stem cell self-renewal by phosphoinositide 3-kinase-dependent signaling. J Biol Chem 2004;279:48063–48070.[Abstract/Free Full Text]

  15. Watanabe S, Umehara H, Murayama K et al. Activation of Akt signaling is sufficient to maintain pluripotency in mouse and primate embryonic stem cells. Oncogene 2006;25:2697–2707.[CrossRef][Medline]

  16. Boiani M, Scholer HR. Regulatory networks in embryo-derived pluripotent stem cells. Nat Rev Mol Cell Biol 2005;6:872–884.[CrossRef][Medline]

  17. Niwa H. How is pluripotency determined and maintained? Development 2007;134:635–646.[Abstract/Free Full Text]

  18. Murayama K, Kimura T, Tarutani M et al. Akt activation induces epidermal hyperplasia and proliferation of epidermal progenitors. Oncogene 2007;26:4882–4888.[CrossRef][Medline]

  19. Solter D, Knowles BB. Immunosurgery of mouse blastocyst. Proc Natl Acad Sci U S A 1975;72:5099–5102.[Abstract/Free Full Text]

  20. Nakano T, Kodama H, Honjo T. In vitro development of primitive and definitive erythrocytes from different precursors. Science 1996;272:722–724.[Abstract]

  21. Kimura T, Ito C, Watanabe S et al. Mouse Germ cell-less as an essential component for nuclear integrity. Mol Cell Biol 2003;23:1304–1315.[Abstract/Free Full Text]

  22. Meijer L, Skaltsounis AL, Magiatis P et al. GSK-3-selective inhibitors derived from Tyrian purple indirubins. Chem Biol 2003;10:1255–1266.[CrossRef][Medline]

  23. Noben-Trauth N, Kohler G, Burki K et al. Efficient targeting of the IL-4 gene in a BALB/c embryonic stem cell line. Transgenic Res 1996;5:487–491.[CrossRef][Medline]

  24. Doble BW, Woodgett JR. GSK-3: Tricks of the trade for a multi-tasking kinase. J Cell Sci 2003;116:1175–1186.[Abstract/Free Full Text]

  25. Jope RS, Johnson GV. The glamour and gloom of glycogen synthase kinase-3. Trends Biochem Sci 2004;29:95–102.[CrossRef][Medline]

  26. Ahn S, Joyner AL. In vivo analysis of quiescent adult neural stem cells responding to Sonic hedgehog. Nature 2005;437:894–897.[CrossRef][Medline]

  27. Androutsellis-Theotokis A, Leker RR, Soldner F et al. Notch signalling regulates stem cell numbers in vitro and in vivo. Nature 2006;442:823–826.[CrossRef][Medline]

  28. Clement V, Sanchez P, de Tribolet N et al. HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr Biol 2007;17:165–172.[CrossRef][Medline]

  29. Karhadkar SS, Bova GS, Abdallah N et al. Hedgehog signalling in prostate regeneration, neoplasia and metastasis. Nature 2004;431:707–712.[CrossRef][Medline]

  30. Varnum-Finney B, Xu L, Brashem-Stein C et al. Pluripotent, cytokine-dependent, hematopoietic stem cells are immortalized by constitutive Notch1 signaling. Nat Med 2000;6:1278–1281.[CrossRef][Medline]

  31. Cartwright P, McLean C, Sheppard A et al. LIF/STAT3 controls ES cell self-renewal and pluripotency by a Myc-dependent mechanism. Development 2005;132:885–896.[Abstract/Free Full Text]

  32. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–676.[CrossRef][Medline]




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