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Cell cycle synchronization of embryonic stem cells: Effect of serum deprivation on the differentiation of embryonic bodies in vitro
BBRC Biochemical and Biophysical Research Communications 333 (2005) 1171–1177 www.elsevier.com/locate/ybbrc
Cell cycle synchronization of embryonic stem cells: Effect of serum deprivation on the differentiation of embryonic bodies in vitro Enming Zhang, Xiaolong Li, Shufang Zhang, Liangqiang Chen, Xiaoxiang Zheng * Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, PR China Received 7 May 2005 Available online 15 June 2005
Abstract Research on stem-cell transplantation has indicated that the success of transplantation largely depends on synchronizing donor cells into the G0/G1 phase. In this study, we investigated the profile of embryonic stem (ES) cell synchronization and its effect on the formation of embryonic bodies (EBs) using cell culture with serum deprivation. The D3 cell line of ES cells was used, and parameters such as cell proliferation and activity, EB formation, and expression of stage-specific embryonic antigen-1 and Oct-4 were investigated. Results showed that the percentage of G0/G1 stage in serum deprivation culture is significantly higher than that in culture with serum supplementation. Synchronized ES cells can reenter the normal cell cycle successfully after serum supply. EBs formed from synchronized ES cells have higher totipotency capability to differentiate into functional neuronal cells than EBs formed from unsynchronized ES cells. Our study provides a method for ES treatment before cell transplantation that possibly helps to decrease the rate of cell death after transplantation. 2005 Published by Elsevier Inc. Keywords: Cell synchronization; Differentiation; Embryonic bodies; Embryonic stem cells; Serum deprivation
Recent studies have shown that embryonic stem (ES) cells could be a cell source for transplantation therapy for nervous system disease such as ParkinsonÕs disease, stroke, and spinal cord injury [1–5]. Normally, two transplantation strategies are applied, as follows: (i) suspended embryonic bodies (EBs) developed from ES cells differentiate further into neuronal precursor cells, followed by transplantation of the precursor cells; and (ii) the EBs or ES cells are transplanted directly into the organism. In these protocols, the differentiational capacity of EBs plays an important role in the transplantation. The status of EB development will decide whether these ES cells can successfully grow and further differentiate into functional target cells in vivo.
*
Corresponding author. Fax: +86 571 8795 1676. E-mail address: [email protected] (X. Zheng).
0006-291X/$ - see front matter 2005 Published by Elsevier Inc. doi:10.1016/j.bbrc.2005.05.200
To acclimatize to the environment of the host, ES cells before transplantation are cultured in sterile medium containing a low concentration of serum. However, the effect of jejune culture treatment on ES cell cycle and differentiation has not been resolved. Furthermore, with stimulation of the mitotic signal, ES cells move from the G0 to the G1 stage. Otherwise, cells undergo differentiation or apoptosis. The G0 phase is a specific stem-cell phase that allows them to retain their ability to engraft as they reenter the cell cycle on the application of stimuli [6,7]. Therefore, it is possible that the differentiation of ES cells is controlled by the proportion of cells in G0 phase over the whole cell cycle. In our studies, we characterized the profile of the cell cycle of the D3 mouse ES cell line by treatment with serum deprivation, and analyzed the effects of synchronization on cell proliferation after EB formation. The results of the present studies are twofold: (i) most of the ES cells can be synchronized in G0/G1 phase by treatment with
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serum deprivation; and (ii) synchronized ES cells can form EBs, which have much greater capacity to differentiate into functional cells than unsynchronized ES cells.
Methods and materials ES cell culture. ES cells of the D3 line were maintained in an undifferentiated state without feeder layer cells and in the presence of mouse leukemia inhibitory factor (LIF). ES cells were cultured at 37 C under 5.0% CO2 in ES culture medium [DulbeccoÕs modified Eagle’s medium (DMEM) supplemented with 0.1 mM b-mercaptoethanol, 15% fetal calf serum (FCS) (Sijiqing, Hangzhou), 0.1 mg ml 1 penicillin–streptomycin, and 1000 U ml 1 LIF]. The medium was replaced every 2 days. Cell cycle synchronization. Briefly, ES cells were cultured to a concentration of no less than 1 · 106 cells ml 1 in ES culture medium. For serum deprivation experiments, cells were washed with DHankÕs solution, and then cultured in different concentrations of FCS and 10 ng ml 1 basic fibroblast growth factor (bFGF; Boehringer–Mannheim, Mannheim, Germany) at different times from 24 to 48 h. Cell cycle analysis. Cellular DNA content was determined by staining cells with propidium iodide and measuring fluorescence by fluorescence-activated cell sorting (FACS; Becton–Dickinson) [8]. ES cells were trypsinized and fixed in cooled 70% ethanol (4 C). The cells were then incubated in a solution containing 1 mg ml 1 RNase and 20 mg ml 1 propidium iodide for 30 min. Subsequently, the cells were transferred into D-HankÕs solution. For each cell population, approximately 10,000 cells were analyzed by FACS, and the proportion of cells in G0/G1, S, and G2/M phases was estimated by the Modfit cell-cycle analysis program. EB formation and differentiation culture. ES cells (25 ll per drop; 5 · 105 cells ml 1) were plated inside the lid of bacterial-grade Petri dishes in standard ES culture medium containing 20% FCS without LIF. Purified water was added to the dishes and the lids were gently turned over to cover the dishes. After 2 days of culture, the cells were replanted on 3.5-cm dishes coated with agar of the same medium. After 2 days of suspension culture, cell aggregates (EBs) were collected and replanted in gelatin-coated 96-well culture dishes (approximately 1–2 EBs per well) in ES culture medium containing 1 lM all-trans retinoic acid (RA; Sigma, St. Louis, MO, USA). The nerve-like cells were counted per EB after RA treatment for 4 days. MTT assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay was performed as previously described [9]. Briefly, cells were produced using the culture methods described above in six-well plates. The cells were then centrifuged at 1000 rpm for 10 min after trypsinization and the supernatant was gently removed. Then 500 ll of DMEM containing 1 mg ml 1 MTT was added to each well. After incubation at 37 C in 5% CO2 for 4 h, the cells were centrifuged at 1000 rpm for 10 min and the supernatant was carefully collected. Dark blue crystals were dissolved by adding 200 ll of isopropyl alcohol/0.4 N HCl to each well. The optical density (OD) was measured on a UV–visible spectrometer (Agilent) using a test wavelength of 570 nm and a reference wavelength of 630 nm. The percentage was calculated using the equation: cell activity (%) = [(ODtest cell ODcontrol cell)/ODcontrol cell] · 100 control cell was not treated with MTT. BrdU incorporation. Cells were seeded at a density of 3 · 105 cells ml 1 and were grown for 24 h in ES culture medium. Cells were then washed once with D-HankÕs solution (pH 7.4) and subsequently starved for 24 h. Bromodeoxyuridine (BrdU) was added to the culture medium to give a final concentration of 10 mM for 20 h. To determine BrdU incorporation, the cells were fixed in cold (4 C) 70% ethanol for 30 min, washed with D-HankÕs solution, 0.2% Triton
X-100, and 2% bovine serum albumin (BSA), and then incubated for 1 h in a monoclonal antibody against BrdU labeled with fluorescein isothiocyanate (FITC; Caltag). After three washes with D-HankÕs solution, the fluorescence intensity of cells was measured using FACS. Immunofluorescence assay. EBs were frozen, sliced (20 lm), and mounted on slides. The resulting in vitro cell monolayers were washed with D-HankÕs solution after the medium was moved. The slides were then fixed with 4% paraformaldehyde for 30 min, washed with D-HankÕs solution containing 0.2% Triton X-100 and were blocked with D-HankÕs solution containing 2% BSA. Cell aggregates were incubated for 1 h with an FITC-conjugated monoclonal antibody against BrdU and rabbit anti-mouse NF-M (Neurofilament M) antibody (1:500) (Chemicon) and goat anti-rabbit IgG1 conjugated with FITC (Chemicon). The fluorescence intensity was measured by confocal laser scanning microscopy (CLSM; Zeiss LSM 10). Data represent the mean values from three independent experiments. Stage-specific embryonic antigen-1 expression assay. An antistage-specific embryonic antigen (SSEA)-1 antibody was used for immunofluorescence measurements on a flow cytometer to analyze the differentiation states of ES cells at different cell cycle stages. Briefly, cells were washed three times with phosphate-buffered saline (PBS) and trypsinized from the plates. Then cells were collected by centrifugation at 1200 rpm and resuspended in 70% alcohol to fix for 40 min. The cells were incubated for 1 h with the first antibodies against SSEA-1 (1:100) (Chemicon), and gently washed three times with PBS. The cells were then incubated for 30–45 min with a species-specific second antibody, FITC-labeled goat anti-mouse IgG1 (Chemicon). After three washes with PBS, cells were analyzed by FACS. Forward scatter (FSC) and side scatter (SSC) were collected in linear mode and FL1 and FL2 in log mode. At least 10,000 cells were collected for each sample and data were analyzed with the CELLQUEST software. RT-PCR assay. Total RNA was isolated from ES cells cultured in vitro using a column and Trizol kit (Sangon, Shanghai, China). A 0.5-lg sample of total RNA was used as a template in a total volume of 20 ll. RT-PCR were carried out using a one-step RT-PCR kit (Sangon) in an Eppendorf Mastercycler as follows: 25 C for 10 min, 42 C for 1 h, and heat denaturation at 94 C for 10 min; then reactions were performed at 94 C for 1 min, followed by 30 cycles of 30 s at 94 C, 30 s at 60 C, and 1 min at 72 C. The program was finished by a 5-min extension at 72 C. The primers used are specific for 300 bp of Oct-4 (forward: 5-GGCGTTCTCTTTGGAAAGGTGTTC-3; reverse: 5-CTCGAACCACATCCTTCTCT-3) and 200 bp of b-actin (forward: 5-CGCACCCACTGGCATTGTCAT-3; reverse: 5-TTCTC CTTGATGTCACGCAC-3). The PCR products were separated by electrophoresis in 1.5% agarose gel in Tris–borate–EDTA (TBE) buffer containing 50 lg ml 1 ethidium bromide. Statistics. Statistical analyses were performed using Microsoft EXCEL. Results are expressed as means ± SEM obtained from at least three observations. Differences between treatments were determined using StudentÕs t test and were judged to be significant at P < 0.05 or P < 0.01.
Results ES cells were synchronized by serum deprivation in vitro The cell cycle of ES cells was recorded over 48 h. ES cell divisions ceased and numerous ES cell deaths were observed in 0.5% FCS culture conditions. After 48 h, 72.2% of serum-deprived ES cells were in G0/G1 phase and 25% were in S and G2/M phases (Fig. 1B). In contrast, 56% of unsynchronized ES cells were in G0/G1 phase and approximately 40% were in S and G2/M
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Fig. 2. Proliferation of mouse ES cells at different stages with FCS starvation treatment. Cells were grown in the medium containing 15% FCS or serum-deprived medium. Incorporated BrdU was detected by FACS. An anti-BrdU antibody was conjugated with FITC. **P < 0.01 values significantly differed from cells cultured in 15% FCS medium. Data represent the means ± SD (n = 3).
of BrdU-positive ES cells decreased to 68%, which was higher than for serum-deprived cells. Reentry of arrested ES cells into the cell cycle
Fig. 1. Effect of serum deprivation on the cell cycle in mouse ES cells. Percentage of ES cells at G0/G1, S, and G2/M stages of the cell cycle cultured in (A) medium containing 15% FCS; and (B) serum-deprived medium (0.5% FCS). ES cells were cultured for 0, 24, 36, and 48 h, and then the samples collected were measured by FACS. Each bar represents the mean ± SD of three independent experiments. (C) The effect of different FCS concentrations on ES cell G0/G1 stage and cell activity after synchronization for 48 h. ES cell survival ability was measured using the MTT method. *P < 0.05, **P < 0.01 values significantly differed from the percentage of cells cultured in 15% FCS medium. Data represent the means ± SD (n = 3).
To investigate the ability of synchronized ES cells to reenter the cell cycle, DNA synthesis released from blocked cells was measured by FACS in 15% FCS medium. The results show that ES cells could be released from their blocked state and mitotic cells were detected in the time course (Fig. 3A). At time zero (0 h), the percentage of cells in G0/G1, S, and G2/M
phases (Fig. 1A). Comparison with control cells (0 h), cell activity of synchronized ES cells decrease to 29% after 48 h. Furthermore, the decrease in FCS concentration increased the number of cells in G1/G0 phase, and decreased the cell activity of cells at the same time (Fig. 1C). Proliferation of ES cells after serum deprivations in vitro To characterize accurately the proliferation of ES cells after serum deprivation and the minimum time of serum deprivation required to arrest the ES cell cycle, further, cells were analyzed quantitatively by the method of BrdU incorporation (Fig. 2). Results show that serum starvation led to a rapid decrease in dividing ES cells. Culture in serum-deprived medium (0.5% FCS) for 48 h decreased the percentage of BrdU-labeled ES cells from 95% to 5%. In contrast, after 48 h the percentage
Fig. 3. Reentry of synchronized ES cells into the cell cycle. (A) After cell cycle synchronization was achieved by serum deprivation, the percentage of DNA-synthesizing cells was recorded at different time points under normal ES culture conditions. Data were collected from three independent experiments. (B) Cell count of DNA-synthesizing cells at different time points after release from cell cycle arrest. Data represent the means ± SD (n = 3). **P < 0.01 values significantly differed from unsynchronized cells with StudentÕs t test.
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phases was 47%, 19%, and 1.7%, respectively. After 48 h, the percentage in G0/G1, S and G2/M phases changed to 14%, 48%, and 12%, respectively. Furthermore, over a period of 5 days, reentered ES cells followed a normal growth curve; however, this curve differed significantly from unsynchronized ES cells after 2 days (Fig. 3B). Effects of ES cell synchronization on EB activity To investigate the EB formation ability of synchronized ES cells, serum-deprived cells were aggregated in 20% FCS culture medium. Compared with unsynchronized ES cells, EB aggregates from serum-deprived cells were small, unequal, and rough (data not shown). Using BrdU incorporation, the proliferation ability of EBs was measured with FACS (Fig. 4A). During the time course monitored, the percentage of BrdU-positive EBs gradually decreased in the serum-starved culture medium. After 48 h of culture, BrdU-positive cells decreased to 0.2%. Moreover, EB aggregates from synchronized cells showed higher fluorescence intensity under CLSM compared to EBs formed from unsynchronized cells (Figs. 4B–E). The fringe of EBs
formed from unsynchronized cells was smooth (Figs. 4B and D) and had a strong cell activity; EBs formed from unsynchronized had an accidented fringe and decrease cell activity (Figs. 4C and E). Changes in EB differentiation capacity after serum deprivation To evaluate SSEA-1 expression in serum-deprived culture medium, ES cells were collected and measured using anti-SSEA-1 antibodies with FACS (Figs. 5A and B). After 48 h of serum starvation, the percentage of SSEA-1 positive cells was 44% and the percentage of positive control cells was 47.8%. After cells were aggregated to form EBs, the percentage of SSEA-1 positive cells was 30.9% for serum-starved cells and 25.7% for control EBs (15% FCS). These results show that the levels of SSEA-1 antigen were significantly higher for synchronized cells than for unsynchronized ES cells 2 days after EB formation. To monitor the effects of cell cycle synchronization on differentiation at the molecular level, we investigated the expression of the transcription factor Oct-4 via RT-PCR assay (Fig. 5C). Using the housekeeping b-actin gene as a
Fig. 4. Effect of ES cell synchronization on EB formation. Synchronized ES cells were allowed to proliferate in drop culture for 2 days and were then replanted in dishes coated with agar. After suspension culture for another 2 days, cell aggregates were collected and replanted in gelatin-coated 24well dishes in ES culture medium containing 10 6 M all-trans RA. (A) Percentage of BrdU-positive EBs in different culture conditions. Data represent the means ± SD (n = 3). **P < 0.01 values significantly differed from EBs derived from unsynchronized cells with StudentÕs t test. (B,D) EBs aggregated from ES cells cultured in 15% FCS medium. (C,E) EBs aggregated from ES cells cultured in 0.5% FCS medium. The cells were stained with anti-BrdU antibody; positive cells are labeled with FITC.
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Fig. 5. Differentiation capacity of EBs from synchronized ES cells and of synchronized cells. SSEA-1 expression in (A) ES cells before EB formation; and (B) EBs from serum deprivation medium and serum-supplemented medium. Cells were collected and then measured by FACS. SSEA-1 expression was detected with anti-SSEA-1 antibodies labeled with FITC. Data represent the means ± SD (n = 3). **P < 0.01 values significantly differed from EBs derived from unsynchronized cells with StudentÕs t test. (C) Oct-4 (300 bp) expression was examined by PT-PCR using b-actin (200 bp) as the standard. Lane 1, EBs from ES cells in serum-deprived culture for 48 h; lane 2, EBs from ES cells in serum-sufficient culture; lane 3, ES cells in serum-deprived culture for 48 h before EB formation; lane 4, ES cells in serum-sufficient culture before EB formation.
control, results showed that expression was higher in EBs formed from synchronized ES cells than from unsynchronized ES cells after synchronization for 48 h. EB directional differentiation into neuronal cells EB differentiation into functional neuronal cells was induced by culture in RA 4/4+ medium. This method of differentiation has been successfully applied to D3 cell
lines. EB survival was analyzed after replanting in culture medium containing 1 lM all-trans RA. Through differentiating culture, the EB survival rate was measured after RA treatment for 2 days. The survival of EBs aggregated from synchronized ES cells was less significant than for unsynchronized ES cells. After 48 h this rate decreased to 0.3, while the rate for control cells was 1 (Fig. 6A). Neuron-like cells extended from the EBs after 4 days of culture. Functional neurons were stained using anti-NF-M antibodies and counted on days 4, 8,
Fig. 6. Synchronization effect on EB differentiation after RA treatment. (A) The EB survival rate was obtained by counting surviving EBs after culture in 4/4+ RA differentiation medium. EB survival rate = number surviving EBs/number of total EBs. (B) Neuronal cells derived from synchronized ES cells were labeled by anti-NF-M antibody after 12 days of differentiation. (C) Control cells (derived from unsynchronized ES cells) were labeled by anti-NF-M antibody after 12 days of differentiation. (D) The number of neuronal cells per EB were counted under the microscope 4–16 days after RA treatment and expressed as mean ± SD. Neuronal cells were calculated with both positive-NF-M cells and axon length exceed 5-fold of soma diameter. All data represent the means ± SD (n = 3). **P < 0.01 values significantly differed from unsynchronized cells with StudentÕs t test.
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12, and 16 (Figs. 6B–D). Results show that the production of neuronal cells was higher for EBs from synchronized ES cells than from unsynchronized cells.
Discussion The transition of ES cells from G0 phase into G1 phase is related to cell fate, such as differentiation, proliferation or apoptosis. It is essential for further differentiation that a large percentage of ES cells in G0 phase develop into EBs. Reports have shown that the development of stem cells is determined by the percentage of cells in G0 phase over the whole cell cycle [10,11]. Serum deprivation is a method commonly used to synchronize cell lines into the G0 phase [12,13]. Our data indicate that most ES cells could be arrested in the G0/G1 phase using serum deprivation culture. Furthermore, synchronized ES cells could reenter a normal cell cycle successfully after serum was resupplied. EBs formed from synchronized ES cells had higher totipotency capability to differentiate into functional cells than those from unsynchronized ES cells. Given the apparent fluctuation of EBs with G0 phase arrest, these results have important implications for ES transplantation and therapy strategies. Our study demonstrates that serum deprivation can affect the ES cell cycle state. Using serum deprivation treatment for 48 h, approximately 72.2% of ES cells were arrested in the G0/G1 stage. Recent analysis by flow cytometry showed similar synchronization efficiencies in the G0/G1 stage after serum starvation. In these experiments, we found that G0/G1 cell numbers increased with decreasing FCS concentration. Meanwhile, the survival ability of ES cells decreased (Fig. 1C). There are two possible explanations for this decrease in cell survival. First, the serum deprivation treatment had a deleterious effect on the growth of synchronized ES cells. Some reports have shown that a decrease in cell survival ability was related to apoptosis induced by DNA fragmentation [14–16]. Second, a large percentage of ES cells in the G0 phase are in a quiescent state, in which cells demonstrate weak metabolism. The question as to whether the starvation treatment induced a decrease in ES cell survival requires further investigation. However, when synchronized ES cells reentered the cell cycle, cell growth curves showed normal changes similar to unsynchronized ES cells over time in the medium containing 15% FCS (Fig. 3). Therefore, even if serum deprivation treatment had a deleterious effect on the cells, ES cells could continue to grow and develop into EBs. One interesting result showed that SSEA-1 expression of synchronized ES cells was significantly higher than that of unsynchronized cells after EB formation. However, SSEA-1 expression of synchronized ES cells was lower than that of unsynchronized cells before EB for-
mation (Fig. 5). The proportion of totipotent cells in EBs can be recorded with the surface antigen SSEA-1. SSEA-1 was first identified as a marker of totipotency in ES cells as detected on blastomeres of 8-cell embryos [17]. This result was also supported by the high expression of Oct-4, a transcription factor which is highly expressed in undifferentiated ES, embryonal carcinoma (EC), and embryonic germ (EG) cells in vitro. Reports have shown that Oct-4 plays an essential role in the establishment and maintenance of toti/pluripotent cells [18]. Our results suggest that EBs from synchronized ES cells had a much greater capacity for further differentiation. On the other hand, this result also proves that the proportion ES cells in the G0 phase strongly affected the differentiation capacity. We consider that G0 phase cells were regularly distributed in the endoblast, mesoblast, and ectoderm of EBs. These G0 phase cells in different positions of the EBs would then be readily available to growth factor. Therefore, we consider that EB G0-phase cells can exert an intrinsic influence on EB differentiation and determine the ES cell position where differentiation begins; growth factors can exert an extrinsic influence on EB differentiation and determine what type of functional cells are differentiated. Our data also indicate that synchronized ES cells could be differentiated into neuronal cells after EBs were replanted in RA 4/4+ differentiation medium. EB from synchronized ES cells had a higher capacity to differentiate into functional neuronal cells than that of unsynchronized ES cells after culturing for 8 days, whereas EBs had a lower survival rate after replanting in RA 4/4+ differentiation medium; the reason for this is unclear. The results demonstrate that serum-starved synchronization treatment has a positive influence on EB differentiation to neuronal cells. Recent reports showed that sterile serum-starvation culture before transplantation led to graft production of dopaminergic neurons or oligodendrocytes after ES cells were transplanted into murine brain, and subsequent transplantation also produced large numbers of teratocarcinoma cells. It was considered that serum deprivation treatment had broken the cell proliferation cycle, which was similar to results for EBs transplanted into a host under specific growth conditions. The serum-starved environment was even similar to the transplant environment, in contrast to serum-supplemented medium. Perhaps serum starvation treatment promotes ES cell differentiation into target cells in the host before cell transplantation. The question as to whether the high rate of teratocarcinoma cells or graft death is related to the culture conditions still requires further investigation. Serum starvation synchronization treatment will allow ES cells to undergo jejune culture at an earlier transplantation stage. Moreover, using this method it is possible to decrease the deleterious effect of jejune culture on transplant cells.
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Acknowledgments This work was supported by: (i) the Zhejiang Provincial Key Laboratory for Chinese Medicine Screening, Exploitation and Medicinal Effectiveness Appraisal of Cardio-cerebral, Vascular and Nervous Systems; and (ii) the Key Laboratory for Biomedical Engineering of the Ministry of Education, PR China. References [1] H.Q. Xian, E. McNichols, A. St Clair, D.I. Gottlieb, A subset of ES-cell-derived neural cells marked by gene targeting, Stem Cells 21 (2003) 41–49. [2] S. Liu, Y. Qu, T.J. Stewart, M.J. Howard, S. Chakrabortty, T.F. Holekamp, J.W. McDonald, Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation, Proc. Natl. Acad. Sci. USA 97 (2000) 6126–6131. [3] Y. Benninger, S. Marino, R. Hardegger, C. Weissmann, A. Aguzzi, S. Brandner, Differentiation and histological analysis of embryonic stem cell-derived neural transplants in mice, Brain Pathol. 10 (2000) 330–341. [4] L.M. Bjorklund, R. Sanchez-Pernaute, S. Chung, T. Andersson, I.Y. Chen, K.S. McNaught, A.L. Brownell, B.G. Jenkins, C. Wahlestedt, K.S. Kim, O. Isacson, From the cover: embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model, Proc. Natl. Acad. Sci. USA 99 (2002) 2344–2349. [5] J.H. Kim, J.M. Auerbach, J.A. Rodriguez-Gomez, I. Velasco, D. Gavin, N. Lumelsky, S.H. Lee, J. Nguyen, R. Sanchez-Pernaute, K. Bankiewicz, R. McKay, Dopamine neurons derived from embryonic stem cells function in an animal model of ParkinsonÕs disease, Nature 418 (2002) 50–56. [6] I. Wilmut, A.E. Schnieke, J. McWhir, A.J. Kind, K.H.S. Campbell, Viable offspring derived from fetal and adult mammalian cells, Nature 385 (1997) 810–813.
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Cell cycle synchronization of embryonic stem cells: Effect of serum deprivation on the differentiation of embryonic bodies in vitro Enming Zhang, Xiaolong Li, Shufang Zhang, Liangqiang Chen, Xiaoxiang Zheng * Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, PR China Received 7 May 2005 Available online 15 June 2005
Abstract Research on stem-cell transplantation has indicated that the success of transplantation largely depends on synchronizing donor cells into the G0/G1 phase. In this study, we investigated the profile of embryonic stem (ES) cell synchronization and its effect on the formation of embryonic bodies (EBs) using cell culture with serum deprivation. The D3 cell line of ES cells was used, and parameters such as cell proliferation and activity, EB formation, and expression of stage-specific embryonic antigen-1 and Oct-4 were investigated. Results showed that the percentage of G0/G1 stage in serum deprivation culture is significantly higher than that in culture with serum supplementation. Synchronized ES cells can reenter the normal cell cycle successfully after serum supply. EBs formed from synchronized ES cells have higher totipotency capability to differentiate into functional neuronal cells than EBs formed from unsynchronized ES cells. Our study provides a method for ES treatment before cell transplantation that possibly helps to decrease the rate of cell death after transplantation. 2005 Published by Elsevier Inc. Keywords: Cell synchronization; Differentiation; Embryonic bodies; Embryonic stem cells; Serum deprivation
Recent studies have shown that embryonic stem (ES) cells could be a cell source for transplantation therapy for nervous system disease such as ParkinsonÕs disease, stroke, and spinal cord injury [1–5]. Normally, two transplantation strategies are applied, as follows: (i) suspended embryonic bodies (EBs) developed from ES cells differentiate further into neuronal precursor cells, followed by transplantation of the precursor cells; and (ii) the EBs or ES cells are transplanted directly into the organism. In these protocols, the differentiational capacity of EBs plays an important role in the transplantation. The status of EB development will decide whether these ES cells can successfully grow and further differentiate into functional target cells in vivo.
*
Corresponding author. Fax: +86 571 8795 1676. E-mail address: [email protected] (X. Zheng).
0006-291X/$ - see front matter 2005 Published by Elsevier Inc. doi:10.1016/j.bbrc.2005.05.200
To acclimatize to the environment of the host, ES cells before transplantation are cultured in sterile medium containing a low concentration of serum. However, the effect of jejune culture treatment on ES cell cycle and differentiation has not been resolved. Furthermore, with stimulation of the mitotic signal, ES cells move from the G0 to the G1 stage. Otherwise, cells undergo differentiation or apoptosis. The G0 phase is a specific stem-cell phase that allows them to retain their ability to engraft as they reenter the cell cycle on the application of stimuli [6,7]. Therefore, it is possible that the differentiation of ES cells is controlled by the proportion of cells in G0 phase over the whole cell cycle. In our studies, we characterized the profile of the cell cycle of the D3 mouse ES cell line by treatment with serum deprivation, and analyzed the effects of synchronization on cell proliferation after EB formation. The results of the present studies are twofold: (i) most of the ES cells can be synchronized in G0/G1 phase by treatment with
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serum deprivation; and (ii) synchronized ES cells can form EBs, which have much greater capacity to differentiate into functional cells than unsynchronized ES cells.
Methods and materials ES cell culture. ES cells of the D3 line were maintained in an undifferentiated state without feeder layer cells and in the presence of mouse leukemia inhibitory factor (LIF). ES cells were cultured at 37 C under 5.0% CO2 in ES culture medium [DulbeccoÕs modified Eagle’s medium (DMEM) supplemented with 0.1 mM b-mercaptoethanol, 15% fetal calf serum (FCS) (Sijiqing, Hangzhou), 0.1 mg ml 1 penicillin–streptomycin, and 1000 U ml 1 LIF]. The medium was replaced every 2 days. Cell cycle synchronization. Briefly, ES cells were cultured to a concentration of no less than 1 · 106 cells ml 1 in ES culture medium. For serum deprivation experiments, cells were washed with DHankÕs solution, and then cultured in different concentrations of FCS and 10 ng ml 1 basic fibroblast growth factor (bFGF; Boehringer–Mannheim, Mannheim, Germany) at different times from 24 to 48 h. Cell cycle analysis. Cellular DNA content was determined by staining cells with propidium iodide and measuring fluorescence by fluorescence-activated cell sorting (FACS; Becton–Dickinson) [8]. ES cells were trypsinized and fixed in cooled 70% ethanol (4 C). The cells were then incubated in a solution containing 1 mg ml 1 RNase and 20 mg ml 1 propidium iodide for 30 min. Subsequently, the cells were transferred into D-HankÕs solution. For each cell population, approximately 10,000 cells were analyzed by FACS, and the proportion of cells in G0/G1, S, and G2/M phases was estimated by the Modfit cell-cycle analysis program. EB formation and differentiation culture. ES cells (25 ll per drop; 5 · 105 cells ml 1) were plated inside the lid of bacterial-grade Petri dishes in standard ES culture medium containing 20% FCS without LIF. Purified water was added to the dishes and the lids were gently turned over to cover the dishes. After 2 days of culture, the cells were replanted on 3.5-cm dishes coated with agar of the same medium. After 2 days of suspension culture, cell aggregates (EBs) were collected and replanted in gelatin-coated 96-well culture dishes (approximately 1–2 EBs per well) in ES culture medium containing 1 lM all-trans retinoic acid (RA; Sigma, St. Louis, MO, USA). The nerve-like cells were counted per EB after RA treatment for 4 days. MTT assay. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma) assay was performed as previously described [9]. Briefly, cells were produced using the culture methods described above in six-well plates. The cells were then centrifuged at 1000 rpm for 10 min after trypsinization and the supernatant was gently removed. Then 500 ll of DMEM containing 1 mg ml 1 MTT was added to each well. After incubation at 37 C in 5% CO2 for 4 h, the cells were centrifuged at 1000 rpm for 10 min and the supernatant was carefully collected. Dark blue crystals were dissolved by adding 200 ll of isopropyl alcohol/0.4 N HCl to each well. The optical density (OD) was measured on a UV–visible spectrometer (Agilent) using a test wavelength of 570 nm and a reference wavelength of 630 nm. The percentage was calculated using the equation: cell activity (%) = [(ODtest cell ODcontrol cell)/ODcontrol cell] · 100 control cell was not treated with MTT. BrdU incorporation. Cells were seeded at a density of 3 · 105 cells ml 1 and were grown for 24 h in ES culture medium. Cells were then washed once with D-HankÕs solution (pH 7.4) and subsequently starved for 24 h. Bromodeoxyuridine (BrdU) was added to the culture medium to give a final concentration of 10 mM for 20 h. To determine BrdU incorporation, the cells were fixed in cold (4 C) 70% ethanol for 30 min, washed with D-HankÕs solution, 0.2% Triton
X-100, and 2% bovine serum albumin (BSA), and then incubated for 1 h in a monoclonal antibody against BrdU labeled with fluorescein isothiocyanate (FITC; Caltag). After three washes with D-HankÕs solution, the fluorescence intensity of cells was measured using FACS. Immunofluorescence assay. EBs were frozen, sliced (20 lm), and mounted on slides. The resulting in vitro cell monolayers were washed with D-HankÕs solution after the medium was moved. The slides were then fixed with 4% paraformaldehyde for 30 min, washed with D-HankÕs solution containing 0.2% Triton X-100 and were blocked with D-HankÕs solution containing 2% BSA. Cell aggregates were incubated for 1 h with an FITC-conjugated monoclonal antibody against BrdU and rabbit anti-mouse NF-M (Neurofilament M) antibody (1:500) (Chemicon) and goat anti-rabbit IgG1 conjugated with FITC (Chemicon). The fluorescence intensity was measured by confocal laser scanning microscopy (CLSM; Zeiss LSM 10). Data represent the mean values from three independent experiments. Stage-specific embryonic antigen-1 expression assay. An antistage-specific embryonic antigen (SSEA)-1 antibody was used for immunofluorescence measurements on a flow cytometer to analyze the differentiation states of ES cells at different cell cycle stages. Briefly, cells were washed three times with phosphate-buffered saline (PBS) and trypsinized from the plates. Then cells were collected by centrifugation at 1200 rpm and resuspended in 70% alcohol to fix for 40 min. The cells were incubated for 1 h with the first antibodies against SSEA-1 (1:100) (Chemicon), and gently washed three times with PBS. The cells were then incubated for 30–45 min with a species-specific second antibody, FITC-labeled goat anti-mouse IgG1 (Chemicon). After three washes with PBS, cells were analyzed by FACS. Forward scatter (FSC) and side scatter (SSC) were collected in linear mode and FL1 and FL2 in log mode. At least 10,000 cells were collected for each sample and data were analyzed with the CELLQUEST software. RT-PCR assay. Total RNA was isolated from ES cells cultured in vitro using a column and Trizol kit (Sangon, Shanghai, China). A 0.5-lg sample of total RNA was used as a template in a total volume of 20 ll. RT-PCR were carried out using a one-step RT-PCR kit (Sangon) in an Eppendorf Mastercycler as follows: 25 C for 10 min, 42 C for 1 h, and heat denaturation at 94 C for 10 min; then reactions were performed at 94 C for 1 min, followed by 30 cycles of 30 s at 94 C, 30 s at 60 C, and 1 min at 72 C. The program was finished by a 5-min extension at 72 C. The primers used are specific for 300 bp of Oct-4 (forward: 5-GGCGTTCTCTTTGGAAAGGTGTTC-3; reverse: 5-CTCGAACCACATCCTTCTCT-3) and 200 bp of b-actin (forward: 5-CGCACCCACTGGCATTGTCAT-3; reverse: 5-TTCTC CTTGATGTCACGCAC-3). The PCR products were separated by electrophoresis in 1.5% agarose gel in Tris–borate–EDTA (TBE) buffer containing 50 lg ml 1 ethidium bromide. Statistics. Statistical analyses were performed using Microsoft EXCEL. Results are expressed as means ± SEM obtained from at least three observations. Differences between treatments were determined using StudentÕs t test and were judged to be significant at P < 0.05 or P < 0.01.
Results ES cells were synchronized by serum deprivation in vitro The cell cycle of ES cells was recorded over 48 h. ES cell divisions ceased and numerous ES cell deaths were observed in 0.5% FCS culture conditions. After 48 h, 72.2% of serum-deprived ES cells were in G0/G1 phase and 25% were in S and G2/M phases (Fig. 1B). In contrast, 56% of unsynchronized ES cells were in G0/G1 phase and approximately 40% were in S and G2/M
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Fig. 2. Proliferation of mouse ES cells at different stages with FCS starvation treatment. Cells were grown in the medium containing 15% FCS or serum-deprived medium. Incorporated BrdU was detected by FACS. An anti-BrdU antibody was conjugated with FITC. **P < 0.01 values significantly differed from cells cultured in 15% FCS medium. Data represent the means ± SD (n = 3).
of BrdU-positive ES cells decreased to 68%, which was higher than for serum-deprived cells. Reentry of arrested ES cells into the cell cycle
Fig. 1. Effect of serum deprivation on the cell cycle in mouse ES cells. Percentage of ES cells at G0/G1, S, and G2/M stages of the cell cycle cultured in (A) medium containing 15% FCS; and (B) serum-deprived medium (0.5% FCS). ES cells were cultured for 0, 24, 36, and 48 h, and then the samples collected were measured by FACS. Each bar represents the mean ± SD of three independent experiments. (C) The effect of different FCS concentrations on ES cell G0/G1 stage and cell activity after synchronization for 48 h. ES cell survival ability was measured using the MTT method. *P < 0.05, **P < 0.01 values significantly differed from the percentage of cells cultured in 15% FCS medium. Data represent the means ± SD (n = 3).
To investigate the ability of synchronized ES cells to reenter the cell cycle, DNA synthesis released from blocked cells was measured by FACS in 15% FCS medium. The results show that ES cells could be released from their blocked state and mitotic cells were detected in the time course (Fig. 3A). At time zero (0 h), the percentage of cells in G0/G1, S, and G2/M
phases (Fig. 1A). Comparison with control cells (0 h), cell activity of synchronized ES cells decrease to 29% after 48 h. Furthermore, the decrease in FCS concentration increased the number of cells in G1/G0 phase, and decreased the cell activity of cells at the same time (Fig. 1C). Proliferation of ES cells after serum deprivations in vitro To characterize accurately the proliferation of ES cells after serum deprivation and the minimum time of serum deprivation required to arrest the ES cell cycle, further, cells were analyzed quantitatively by the method of BrdU incorporation (Fig. 2). Results show that serum starvation led to a rapid decrease in dividing ES cells. Culture in serum-deprived medium (0.5% FCS) for 48 h decreased the percentage of BrdU-labeled ES cells from 95% to 5%. In contrast, after 48 h the percentage
Fig. 3. Reentry of synchronized ES cells into the cell cycle. (A) After cell cycle synchronization was achieved by serum deprivation, the percentage of DNA-synthesizing cells was recorded at different time points under normal ES culture conditions. Data were collected from three independent experiments. (B) Cell count of DNA-synthesizing cells at different time points after release from cell cycle arrest. Data represent the means ± SD (n = 3). **P < 0.01 values significantly differed from unsynchronized cells with StudentÕs t test.
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phases was 47%, 19%, and 1.7%, respectively. After 48 h, the percentage in G0/G1, S and G2/M phases changed to 14%, 48%, and 12%, respectively. Furthermore, over a period of 5 days, reentered ES cells followed a normal growth curve; however, this curve differed significantly from unsynchronized ES cells after 2 days (Fig. 3B). Effects of ES cell synchronization on EB activity To investigate the EB formation ability of synchronized ES cells, serum-deprived cells were aggregated in 20% FCS culture medium. Compared with unsynchronized ES cells, EB aggregates from serum-deprived cells were small, unequal, and rough (data not shown). Using BrdU incorporation, the proliferation ability of EBs was measured with FACS (Fig. 4A). During the time course monitored, the percentage of BrdU-positive EBs gradually decreased in the serum-starved culture medium. After 48 h of culture, BrdU-positive cells decreased to 0.2%. Moreover, EB aggregates from synchronized cells showed higher fluorescence intensity under CLSM compared to EBs formed from unsynchronized cells (Figs. 4B–E). The fringe of EBs
formed from unsynchronized cells was smooth (Figs. 4B and D) and had a strong cell activity; EBs formed from unsynchronized had an accidented fringe and decrease cell activity (Figs. 4C and E). Changes in EB differentiation capacity after serum deprivation To evaluate SSEA-1 expression in serum-deprived culture medium, ES cells were collected and measured using anti-SSEA-1 antibodies with FACS (Figs. 5A and B). After 48 h of serum starvation, the percentage of SSEA-1 positive cells was 44% and the percentage of positive control cells was 47.8%. After cells were aggregated to form EBs, the percentage of SSEA-1 positive cells was 30.9% for serum-starved cells and 25.7% for control EBs (15% FCS). These results show that the levels of SSEA-1 antigen were significantly higher for synchronized cells than for unsynchronized ES cells 2 days after EB formation. To monitor the effects of cell cycle synchronization on differentiation at the molecular level, we investigated the expression of the transcription factor Oct-4 via RT-PCR assay (Fig. 5C). Using the housekeeping b-actin gene as a
Fig. 4. Effect of ES cell synchronization on EB formation. Synchronized ES cells were allowed to proliferate in drop culture for 2 days and were then replanted in dishes coated with agar. After suspension culture for another 2 days, cell aggregates were collected and replanted in gelatin-coated 24well dishes in ES culture medium containing 10 6 M all-trans RA. (A) Percentage of BrdU-positive EBs in different culture conditions. Data represent the means ± SD (n = 3). **P < 0.01 values significantly differed from EBs derived from unsynchronized cells with StudentÕs t test. (B,D) EBs aggregated from ES cells cultured in 15% FCS medium. (C,E) EBs aggregated from ES cells cultured in 0.5% FCS medium. The cells were stained with anti-BrdU antibody; positive cells are labeled with FITC.
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Fig. 5. Differentiation capacity of EBs from synchronized ES cells and of synchronized cells. SSEA-1 expression in (A) ES cells before EB formation; and (B) EBs from serum deprivation medium and serum-supplemented medium. Cells were collected and then measured by FACS. SSEA-1 expression was detected with anti-SSEA-1 antibodies labeled with FITC. Data represent the means ± SD (n = 3). **P < 0.01 values significantly differed from EBs derived from unsynchronized cells with StudentÕs t test. (C) Oct-4 (300 bp) expression was examined by PT-PCR using b-actin (200 bp) as the standard. Lane 1, EBs from ES cells in serum-deprived culture for 48 h; lane 2, EBs from ES cells in serum-sufficient culture; lane 3, ES cells in serum-deprived culture for 48 h before EB formation; lane 4, ES cells in serum-sufficient culture before EB formation.
control, results showed that expression was higher in EBs formed from synchronized ES cells than from unsynchronized ES cells after synchronization for 48 h. EB directional differentiation into neuronal cells EB differentiation into functional neuronal cells was induced by culture in RA 4/4+ medium. This method of differentiation has been successfully applied to D3 cell
lines. EB survival was analyzed after replanting in culture medium containing 1 lM all-trans RA. Through differentiating culture, the EB survival rate was measured after RA treatment for 2 days. The survival of EBs aggregated from synchronized ES cells was less significant than for unsynchronized ES cells. After 48 h this rate decreased to 0.3, while the rate for control cells was 1 (Fig. 6A). Neuron-like cells extended from the EBs after 4 days of culture. Functional neurons were stained using anti-NF-M antibodies and counted on days 4, 8,
Fig. 6. Synchronization effect on EB differentiation after RA treatment. (A) The EB survival rate was obtained by counting surviving EBs after culture in 4/4+ RA differentiation medium. EB survival rate = number surviving EBs/number of total EBs. (B) Neuronal cells derived from synchronized ES cells were labeled by anti-NF-M antibody after 12 days of differentiation. (C) Control cells (derived from unsynchronized ES cells) were labeled by anti-NF-M antibody after 12 days of differentiation. (D) The number of neuronal cells per EB were counted under the microscope 4–16 days after RA treatment and expressed as mean ± SD. Neuronal cells were calculated with both positive-NF-M cells and axon length exceed 5-fold of soma diameter. All data represent the means ± SD (n = 3). **P < 0.01 values significantly differed from unsynchronized cells with StudentÕs t test.
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12, and 16 (Figs. 6B–D). Results show that the production of neuronal cells was higher for EBs from synchronized ES cells than from unsynchronized cells.
Discussion The transition of ES cells from G0 phase into G1 phase is related to cell fate, such as differentiation, proliferation or apoptosis. It is essential for further differentiation that a large percentage of ES cells in G0 phase develop into EBs. Reports have shown that the development of stem cells is determined by the percentage of cells in G0 phase over the whole cell cycle [10,11]. Serum deprivation is a method commonly used to synchronize cell lines into the G0 phase [12,13]. Our data indicate that most ES cells could be arrested in the G0/G1 phase using serum deprivation culture. Furthermore, synchronized ES cells could reenter a normal cell cycle successfully after serum was resupplied. EBs formed from synchronized ES cells had higher totipotency capability to differentiate into functional cells than those from unsynchronized ES cells. Given the apparent fluctuation of EBs with G0 phase arrest, these results have important implications for ES transplantation and therapy strategies. Our study demonstrates that serum deprivation can affect the ES cell cycle state. Using serum deprivation treatment for 48 h, approximately 72.2% of ES cells were arrested in the G0/G1 stage. Recent analysis by flow cytometry showed similar synchronization efficiencies in the G0/G1 stage after serum starvation. In these experiments, we found that G0/G1 cell numbers increased with decreasing FCS concentration. Meanwhile, the survival ability of ES cells decreased (Fig. 1C). There are two possible explanations for this decrease in cell survival. First, the serum deprivation treatment had a deleterious effect on the growth of synchronized ES cells. Some reports have shown that a decrease in cell survival ability was related to apoptosis induced by DNA fragmentation [14–16]. Second, a large percentage of ES cells in the G0 phase are in a quiescent state, in which cells demonstrate weak metabolism. The question as to whether the starvation treatment induced a decrease in ES cell survival requires further investigation. However, when synchronized ES cells reentered the cell cycle, cell growth curves showed normal changes similar to unsynchronized ES cells over time in the medium containing 15% FCS (Fig. 3). Therefore, even if serum deprivation treatment had a deleterious effect on the cells, ES cells could continue to grow and develop into EBs. One interesting result showed that SSEA-1 expression of synchronized ES cells was significantly higher than that of unsynchronized cells after EB formation. However, SSEA-1 expression of synchronized ES cells was lower than that of unsynchronized cells before EB for-
mation (Fig. 5). The proportion of totipotent cells in EBs can be recorded with the surface antigen SSEA-1. SSEA-1 was first identified as a marker of totipotency in ES cells as detected on blastomeres of 8-cell embryos [17]. This result was also supported by the high expression of Oct-4, a transcription factor which is highly expressed in undifferentiated ES, embryonal carcinoma (EC), and embryonic germ (EG) cells in vitro. Reports have shown that Oct-4 plays an essential role in the establishment and maintenance of toti/pluripotent cells [18]. Our results suggest that EBs from synchronized ES cells had a much greater capacity for further differentiation. On the other hand, this result also proves that the proportion ES cells in the G0 phase strongly affected the differentiation capacity. We consider that G0 phase cells were regularly distributed in the endoblast, mesoblast, and ectoderm of EBs. These G0 phase cells in different positions of the EBs would then be readily available to growth factor. Therefore, we consider that EB G0-phase cells can exert an intrinsic influence on EB differentiation and determine the ES cell position where differentiation begins; growth factors can exert an extrinsic influence on EB differentiation and determine what type of functional cells are differentiated. Our data also indicate that synchronized ES cells could be differentiated into neuronal cells after EBs were replanted in RA 4/4+ differentiation medium. EB from synchronized ES cells had a higher capacity to differentiate into functional neuronal cells than that of unsynchronized ES cells after culturing for 8 days, whereas EBs had a lower survival rate after replanting in RA 4/4+ differentiation medium; the reason for this is unclear. The results demonstrate that serum-starved synchronization treatment has a positive influence on EB differentiation to neuronal cells. Recent reports showed that sterile serum-starvation culture before transplantation led to graft production of dopaminergic neurons or oligodendrocytes after ES cells were transplanted into murine brain, and subsequent transplantation also produced large numbers of teratocarcinoma cells. It was considered that serum deprivation treatment had broken the cell proliferation cycle, which was similar to results for EBs transplanted into a host under specific growth conditions. The serum-starved environment was even similar to the transplant environment, in contrast to serum-supplemented medium. Perhaps serum starvation treatment promotes ES cell differentiation into target cells in the host before cell transplantation. The question as to whether the high rate of teratocarcinoma cells or graft death is related to the culture conditions still requires further investigation. Serum starvation synchronization treatment will allow ES cells to undergo jejune culture at an earlier transplantation stage. Moreover, using this method it is possible to decrease the deleterious effect of jejune culture on transplant cells.
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