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ISSN : 1738-2432(Print)
ISSN : 2288-0151(Online)
Reproductive & developmental Biology Vol.36 No.1 pp.21-26
DOI :

Suspension Culture-Mediated Tetraploid Formation in Mouse Embryonic Stem Cells

Jae Hee Lee1,2,§, Seung Pyo Gong3,§, Jeong Mook Lim1,4,5, Seung Tae Lee6,†
6Department of Animal Biotechnology, Kangwon National University
1Laboratory of Stem Cell and Bioevaluation, WCU Biomodulation Program, Seoul National University, 2Department of Obstetrics, Gynecology & Reproductive Biology, Michigan State University College of Human Medicine, 3Department of Marine Biomaterials and Aquaculture, Pukyong National University, 4Department of Agricultural Biotechnology, Seoul National University, 5Cancer Research Institute, Seoul National University College of Medicine
Received: 22 February 2012, Accepted: 26 March 2012

Abstract

Suspension culture is a useful tool for culturing embryonic stem (ES) cells in large-scale, but the stability of pluripotencyand karyotype has to be maintained in vitro for clinical application. Therefore, we investigated whether thechromosomal abnormality of ES cells was induced in suspension culture or not. The ES cells were cultured in suspensionas a form of aggregate with or without mouse embryonic fibroblasts (MEFs), and 0 or 1,000 U/ml leukemiainhibitory factor (LIF) was treated to suspended ES cells. After culturing ES cells in suspension, their karyotype, DNAcontent, and properties of pluripotency and differentiation were evaluated. As a result, the formation of tetraploid EScell population was significantly increased in suspension culture in which ES cells were co-cultured with both MEFsand LIF. Tetraploid ES cell population was also generated when ES cells were cultured alone in suspension regardlessof the existence of LIF. On the other hand, the formation of tetraploid ES cell population was not detected in LIF-freecondition, in which MEFs were included. The origin of tetraploid ES cell population was turned out to be E14 EScells and not MEFs by microsatellite analysis and the basic properties of them were still maintained despiteploidy-conversion to tetraploidy. Furthermore, we identified the ploidy shift from tetraploidy to near-triploidy astetraploid ES cells were differentiated spontaneously. From these results, we demonstrated that suspension culturesystem could induce ploidy-conversion generating tetraploid ES cell population. Moreover, optimization of suspensionculture system may make possible mass-production of ES cells.

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INTRODUCTION

 The embryonic stem (ES) cells are considered as an important material for cell replacement therapy to the injuries and degenerative diseases due to their unique property having both self-renewal activity and differentiation potential (Lui et al., 2009). For successful clinical implementation using the ES cells, the reproducible culture system for a large-scale expansion of the ES cells is necessarily required (Krawetz et al., 2010). As one of those methods, suspension culture system has shown that the pluripotency of ES cells can be maintained after long-term expansion by combining with bioreactor (Cormier et al., 2006; zur Nieden et al., 2007). In spite of useful properties for suspension culture system, however, the relationship between culture condition and maintenance of chromosomal normality has to be seriously considered because chromosomal abnormality of ES cells might provoke malfunction and tumorigenesis of replaced tissues when applied in real therapy (Blum and Benvenisty, 2009).

 In this study, for examining the effect of suspension culture on chromosome abnormality of the ES cells, the ploidy conversion of the ES cells after culturing as aggregates in suspension culture using various conditions was investigated. Subsequently, the ES cells experiencing ploidy conversion were characterized with diverse parameters.

MATERIALS AND METHODS

Animals

 All of the procedures for animal management, breeding, and surgery followed the standard operation protocols of Seoul National University. The review board of institutional animal care and use committee of Seoul National University approved the use of animals and the relevant experimental procedures (approval no. SNU-070420-5).

Cell Preparation

For the derivation of mouse embryonic fibroblasts (MEFs), 13.5-day post-coitus fetuses of the B6D2F1 (C-57BL/6 X DBA2) and ICR strains were killed, and the visceral organs, heads, and extremities of the fetuses were removed under microscope. Embryonic fibroblasts collected from the remaining tissue were dissociated with 0.25% (v/v) trypsin (Gibco Invitrogen, Grand Island, NY) and the dissociated MEFs was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Invitrogen) supplemented with 15% (v/v) heat-inactivated fetal bovine serum (FBS; HyClone, Logan, UT) and 1% (v/v) mixed solution of penicillin and streptomycin (Gibco Invitrogen). E14 ES cells were purchased from ATCC (Manassas, VA) and culture of E14 ES cells on 10 μg/ml mitomycin C (Chemicon International, Temecula, CA)-inactivated ICR MEFs was conducted in standard mouse ES cell culture medium consisting of DMEM (Gibco Invitrogen) supplemented with 15% (v/v) heat-inactivated FBS (HyClone), 0.1 mM β-mercaptoethanol (Gibco Invitrogen), 1% (v/v) nonessential amino acids (NEAA; Gibco Invitrogen), 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, MO), 2 mM L-glutamine (Gibco Invitrogen), 1% (v/v) mixed solution of penicillin and streptomycin (Gibco Invitrogen) and 1,000 U/ml mouse leukemia inhibitory factor (LIF; Chemicon International). Subpassage of E14 ES cells was conducted every 3 day, with daily exchanging fresh medium during subculture.

Suspension Culture by Aggregate Formation

 For aggregate formation, E14 ES cells and B6D2F1  MEFs without or with treatment of 10 μg/ml mitomycin C (Chemicon International) were mixed in the ratio of nine to one and co-cultured on bacterial-grade petri dish for 7 days in the standard mouse ES cell culture medium. Moreover, for obtaining aggregates without MEFs, only E14 ES cells were suspended for 7 day in the standard mouse ES cell culture medium. Subsequently, formed aggregates were cultivated in standard mouse ES cell culture medium supplemented without or with 1,000 U/ml LIF (Chemicon International) for 14 days.

Adherent Culture of Aggregates after Suspension Culture

 Six aggregates together were dissociated using a mixture of 0.25% (v/v) trypsin (Gibco Invitrogen) and 285 collagenase digestion units (CDU)/ml of collagenase type Ⅳ (Sigma-Aldrich) in the same ratio. Freely dissociated cells were seeded on MEF monolayer derived from the mice of ICR strain and subsequently colony formation was identified. Single colony was picked by glass pipette and seeded on ICR MEF monolayer in 24-multi dishes after trypsinization. The cell lines derived from single colonies were proliferated and used to further experiments.

Karyotyping

 Two millions of cells were washed in Dulbecco’s phosphate buffered saline (DPBS; Gibco Invitrogen) and treated with 0.075 M KCl (Sigma-Aldrich) solution for 10 min in 37℃. The swollen cells were fixed with cold fixative solution comprised of methanol and acetic acid in the ratio of 3:1 and the fixative solution was changed three times by centrifuging at 1,000 rpm for 5 minutes. Metaphase chromosomes were spread onto ethanol-treated slides and stained with Giemsa’s solution containing 10% (v/v) KARYOMAX®  Giemsa stain (Gibco Invitrogen) in Gurr’s buffer. After washing in distilled water, the slides were air-dried and the number of chromosomes was counted.

Short-Tandom Repeat Microsatellite Analysis

 Short-tandom repeat microsatellite analysis was performed with genomic DNA extracted from B6D2F1 and ICR MEFs, E14 ES cells, and the cell lines derived from single colonies. Four specific mouse microsatellite primers (D3Mit200, D4Mit251, D11Mit4, and D15Mit159) were collected from public database provided from Mouse Genome Informatics (http://www.informatics.jax.org). The genomic DNA from the samples was amplified by PCR for four microsatellite loci. The PCR products were subsequently size-fractioned by agarose gel electrophoresis and visualized by ethidium bromide staining.

Characterization of ES Cells by Immunostaining

 The ES cells were fixed with 4% (v/v) formaldehyde (Sigma-Aldrich) at room temperature for 10 min. After washing twice with DPBS, the cells were stained by Dako Cytomation kit (Dako Cytomation, Carpinteria, CA) with anti-Oct-4 (1:500 dilution; BD Biosciences, Franklin Lakes, NJ) antibody. To confirm spontaneous differentiation in-vitro, the colonies were dissociated and subsequently suspended in LIF-free DMEM containing 15% (v/v) FBS (HyClone), 1% (v/v) NEAA (Gibco Invitrogen), 0.1mM β-mercaptoethanol (Gibco Invitrogen), and 1% (v/v) mixed solution of penicillin and streptomycin (Gibco Invtrogen). When embryoid bodies (EBs) were formed after 4 days, 10 EBs were seeded in each well of 4-multi well dishes and differentiated spontaneously cultured for 10 to 14 days. Subsequently, differentiated ES cells was immunostained with anti-S-100 (1:500 dilution; Abcam, Cambridge, MA), anti-alpha-smooth muscle actin (SMA; 1:100 dilution; Abcam), and anti-Troma-1 (1:200 dilution; Developmental Studies Hybridoma Bank) antibodies by using Dako Cytomation kit (Dako Cytomation) according to manufacturer’s instruction.

Analysis of DNA Content by Propidium Iodide (PI) Staining

 The harvested cells were washed in Ca2+- and Mg2+-free DPBS (Gibco Invitrogen) and fixed by suspension in 70% (v/v) ethanol (Sigma-Aldrich) for overnight at 4℃. The fixed cells were rinsed within cold DPBS twice and re-suspended in PI solution containing 50 ug /ml PI (Sigma-Aldrich), 0.1% (v/v) triton X-100 (Sigma-Aldrich), and 0.2 mg/ml RNase A (Roche, Basel, Switzerland). After 10 min at room temperature in dark, the cells were analyzed by BD FACS CaliburTM Flow Cytometer (Becton Dickinson, San Jose, CA) and the data was analyzed using the CELL QuestTM software (Becton Dickinson).

Statistical Analysis

 The Statistical Analysis System (SAS) program was used for analyzing statistically the numerical data obtained. When analysis of variance (ANOVA) in the SAS package detected a significant main effect, each value was compared by the least square method. The significant differences among treatment were determined at p<0.05.

RESULTS

The Effects of Suspension Culture under Various Conditions on the Formation Of Tetraploid ES Cells

 In order to identify whether suspension culture can induce ploidy abnormality of the ES cells or not, culture of the E14 ES cells as aggregates was conducted in various suspension culture condition. At 14 days culture, aggregates were dissociated and cultured on ICR MEF monolayer. Randomly selected colonies were singly cultured and single colony-derived cell lines were analyzed on karyotype and DNA contents. As shown in Table 1, total 173 cell lines were established from single colonies derived from suspension culture and 12 cell lines (12/173=6.9%) among them were identified to have tetraploidy (Fig. 1). In contrast, there was no tetraploid ES cell line from adherent culture (0/48=0%). In suspension culture with MEFs, the addition of LIF induced significantly higher frequency of tetraploid-conversion than LIF-free condition (15.4 to 27.3% vs. 0%, p<0.05) regardless of mitotic activity of MEFs. Of those, the condition having mitotically activated MEFs with LIF showed the highest frequency of conversion to tetraploid among the treatment groups (3/11=27.3%). Whereas, the formation of tetraploid ES cell population was not detected when LIF was not added to the condition having mitotically activated MEFs (0/55=0%). In case in which ES cells were cultured alone in suspension, we could identify the formation of tetraploid ES cell population regardless of the existence of LIF (4.3 to 10.4%).

Table 1. Formation of tetraploid embryonic stem (ES) cells in different conditions of suspension culture

Fig. 1. Metaphase spread showing tetraploid chromosome of embryonic stem cells (ESCs). Chromosome spreads on slides were stained with Giemsa solution (A) and cytovision apparatus showed 80 chromosome arrangement (B).

Identification of Cell Origin

 In order to identify whether tetraploid ES cell lines are derived from E14 ES cells or MEFs, short-tandom repeat microsatellite analysis was performed in 12 lines of tetraploid ES cells and E14 ES cells, B6D2F1 MEFs, and ICR MEFs as control samples. As shown in Fig. 2, four microsatellite loci of all 12 tetraploid ES cells were exactly same with those of E14 ES cells, indicating that all tetraploid ES cells were derived from the E14 ES cells cultured in suspension culture system.

Fig. 2. Origin of tetraploid embryonic stem (ES) cells formed after suspension culture in different conditions. Short-tandom repeat microsatellite analysis was performed in E14 ES cells, B6D2F1 mouse embryonic fibroblasts (MEFs), ICR MEFs and tetraploid ES cells. The patterns of four microsatellite makers (D3Mit200, D4Mit251, D11Mit4 and D15Mit159) in tetraploid ES cells were exactly matched with those of E14 ES cells.

Characterization and Ploidy Shift of Tetraploid Es Cells

 Tetraploid ES cells showed high level of Oct-4 expression and underwent in-vitro differentiation to the three germ layer-specific cell types when spontaneous differentiation was induced in LIF-free medium, suggesting that they still possess self-renewal (Fig. 3A) and differentiation potentials (Fig. 3B) of ES cells. In addition, we estimated the DNA contents of tetraploid ES cells spontaneously differentiated for 0, 7, 14, 21 days post-EB formation and the fact that DNA contents of the cells are slightly shifted from tetraploidy to neartriploidy during differentiation progress (Fig. 3C) was identified.

Fig. 3. Fig. 3. Characterization of tetraploid ES cells and detection of ploidy shift. (A) Tetraploid ES cells were characterized by staining Oct-4 as a stem cell-specific marker and showed strong expression of Oct-4. (B) Tetraploid ES cells were spontaneously differentiated for 7 days after formation of embryoid bodies (EBs). Immunocytochemistry of the differentiated EBs was conducted using three germ layer-specific markers of S-100, smooth muscle actin (SMA) and Troma-1 and the differentiated cells comprising EBs were positively stained with one of these markers. (C) FACS analysis was performed to analyze DNA contents of the differentiating tetraploid ES cells. Tetraploid ES cells were spontaneously differentiated and the EBs formed were collected at day 0, 7, 14, and 21. Each EB group was dissociated and analyzed for DNA contents by FACS. Ploidy of tetraploid ES cells was shifted to near-triploidy as differentiation is progressed.

DISCUSSION

 In this study, we suggested that suspension culture of ES cells can generate the chromosome abnormality which may provoke the alteration on cellular properties of ES cells. As the results, we have shown that suspension culture system induces the formation of tetraploid ES cells in high frequency (12 of total 173 cell lines analyzed, 6.9%), whereas this phenomenon could be inhibited in the suspension culture condition that ES cells were cultured with mitotically-active MEFs in the absence of LIF (0 of 55 cell lines analyzed, 0%). Tetraploid ES cells derived from diploid ES cells showed normal properties of ES cells in the expression of Oct-4 protein and in vitro differentiation to three germ layer-specific cell types. Additionally, it was identified that ploidy of tetraploid ES cells could be slightly shifted from tetraploid to near-triploid as the differentiation is progressed.

 Our results support previous reports that the chromosomal instability may appear in ES cells while cultivating them continuously in-vitro (Cowan et al., 2004; Draper et al., 2004; Inzunza et al., 2004; Rosler et al., 2004; Maitra et al., 2005; Mitalipova et al., 2005). In spite of the stemness of tetraploid ES cells, these ES cells cannot be used in clinical application. In fact, the chromosomal mutation leads to aneuploidy and it may be one important step in tumorigenesis (Storchova and Pellman, 2004). Especially, tetraploid cells constitute a metastable intermediate between normal euploid and cancer-associated aneuploid (Rello-Varona et al., 2009). Tetraploidization may arise in tumor cell precursors, through various mechanisms except for cell-to-cell fusion. The chromosome non-disjunction can promote cleavage furrow regression and these cells subsequently have a tetraploid (Shi and King, 2005). The cells also can become tetraploid after arrest prolonged by activation of spindle assembly checkpoint (Musacchio and Salmon, 2007). To overcome this risk, the exact mechanism of karyotype conversion should be revealed.

 Traditionally, LIF and MEFs are necessarily required in the culture of mouse ES cells. LIF is an essential factor supplemented to the culture medium as it plays a key role in maintaining undifferentiated state of ES cells (Chambers, 2004). In case of MEFs, they are usually provided as a feeder cell layer in ES cell culture, through which they support in-vitro maintenance, growth, and differentiation of ES cells. The results of this study show that LIF might be one of factors to be able to induce ploidy-conversion of ES cells in suspension culture system. In suspension culture of ES cells with mitotically-active or –inactive MEFs, the addition of LIF significantly increased the formation rate of tetraploid ES cell population. Our previous study reported that MEFs themselves secret LIF in LIF-free medium (Lee et al., 2009). Thus, simultaneous treatment of LIF and LIFsecreting MEFs can make excess LIF supply in culture, which may contribute to generate tetraploid ES cell population specifically in suspension culture environment that may require different concentration of LIF compared with traditional adherent culture of ES cells. In this viewpoint, to appropriately adjust LIF concentration or the cell number of MEFs might be a determinant to prevent the generation of tetraploid ES cell population in suspension culture system.

 In addition, we observed the ploidy shift of tetraploid ES cells from tetraploidy to near-triploidy during spontaneous differentiation process. This phenomenon implies that the ploidy of ES cells may be artificially controlled in-vitro state. Underlying mechanism to be remained for further investigation might provide an important cue to develop the technology for preventing in-vitro alteration of genetic status in ES cells.

 In conclusion, our results demonstrate that suspension culture of ES cells supplemented with LIF and/or MEFs can induce an abnormality of karyotype. Although suspension-cultured ES cells retain a pluripotency, the conversion of ES cells to tetraploid would be regarded as a serious matter for clinical application. Therefore, we, in this study, suggest that a specific suspension culture condition by using only mitotically-active MEFs with ES cells can make it possible a stable culture without ploidy-conversion of ES cells. Furthermore, this system will be applied as an alternative method for a large-scale expansion of ES cells by optimizing LIF concentration and/or the cell number of MEFs.

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