Enhanced effects of secreted soluble factor preserve better pluripotent state of embryonic stem cell culture in a membrane-based compartmentalized micro-bioreactor
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- Chowdhury, M.M., Katsuda, T., Montagne, K. et al. Biomed Microdevices (2010) 12: 1097. doi:10.1007/s10544-010-9464-8
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Pluripotent stem cells are under the influence of soluble factors in a diffusion dominant in vivo microenvironment. In order to investigate the effects of secreted soluble factors on embryonic stem cell (ESC) behavior in a diffusion dominant microenvironment, we cultured mouse ESCs (mESCs) in a membrane-based two-chambered micro-bioreactor (MB). To avoid disturbing the cellular environment in the top chamber of the MB, only the culture medium of the bottom chamber was exchanged. Cell growth in the MB after 5 days of culture was similar to that in conventional 6-well plate (6-WP) and membrane-based Transwell insert (TW) cultures, indicating adequate nutrient supply in the MB. However, the cells retained higher expression of pluripotency markers (Oct4, Sox2 and Rex1) and secreted soluble factors (FGF4 and BMP4) in the MB. Inhibition of FGF4 activity in the MB and TW resulted in a similar cellular response. However, in contrast to the TW, inhibition of BMP4 activity revealed that autocrine action of the upregulated BMP4, which acted cooperatively with leukemia inhibitory factor (LIF), upregulated the pluripotency markers expression in the MB culture. We propose that BMP4 accumulated in the diffusion dominant microenvironment of the MB upregulated its own expression by a positive feedback mechanism—in contrast to the macro-scale culture systems—thereby enhancing the pluripotency of mESCs. The micro-scale culture platform developed in this study enables the investigation of the effects of soluble factors on ESCs in a diffusion dominant microenvironment, and is expected to be used to modulate the ESC fate choices.
KeywordsEmbryonic stem cellSoluble factorsDiffusionMicroenvironmentMicro-bioreactor
The autocrine and paracrine actions of soluble factors have an important role in directing pluripotent stem cell fate choices in vivo (Gadue et al. 2005; Loebel et al. 2003). Pluripotent stem cells and their progenies remain in a diffusion dominant microenvironment enclosed by the trophectoderm and extra-embryonic part until an appreciable amount of mass flow by convection occurs after the onset of blood circulation (Nagy et al. 2003). At the initial stage of embryo development, the fate of pluripotent cells is influenced by the adequate signaling of soluble factors in the microenvironment. In vitro, ESC fate is also modulated by soluble factors (Kunath et al. 2007; Ying et al. 2003a). Although exogenous soluble factors can be added to the in vitro culture systems to control ESC fate, it is necessary to consider the influence of endogenous soluble factors which are secreted by the cells (Wiles and Proetzel 2006). This is highlighted by the fact that the addition of exogenous soluble factors has little influence on the initial differentiation of embryoid bodies (EBs) but influences the successive maturation of differentiated progenies towards the matured cell types (Ogawa et al. 2005; Wiles and Proetzel 2006). Furthermore, neuronal stem cells can be derived efficiently from mouse ESCs (mESCs) without the addition of exogenous factors (Ying et al. 2003b). Therefore, a culture system which mimics the diffusion dominant nature of the in vivo microenvironment is of great importance in order to improve our understanding of stem cell biology and control the stem cell fate decision (Loebel et al. 2003; Murry and Keller 2008).
Microfluidic technology provides advanced tools to develop micro-scale culture systems in an in vivo relevant dimension as well as to control mass transfer modes in the cellular microenvironment (Meyvantsson and Beebe 2008). Various micro-scale culture systems have been developed for ESC culture, but little is known about the effects of secreted soluble factors in these systems. Moreover, before proceeding to the differentiation of ESCs, it is necessary to characterize the differences between the micro and macro-scale cultures, namely regarding the effects of cell secreted soluble factors on ESC behavior. Human ESCs (hESCs) cells were cultured in straight micro-channels in static (Abhyankar et al. 2003) and semi-static (Korin et al. 2009) conditions. Although these cultures facilitated the accumulation of soluble factors around the cells owing to the diffusion dominant nature of static micro-scale culture, their effects on the cells were not investigated. Furthermore, the environment changed abruptly because of the daily replacement of the total culture medium. In micro-fabricated wells, hESCs were found to remain undifferentiated for more than two weeks (Mohr et al. 2006). The reason for that was not identified, but most likely resulted from soluble factors, cell-cell contacts and the extra-cellular matrix (ECM) produced by the cells. Some studies focused on controlling ESC microenvironment using perfusion-based systems (Figallo et al. 2007; Kim et al. 2006). In one of those studies, mESCs were cultured in microfluidic arrays at different flow rates, and the cell colonies showed flow-dependent size variations (Kim et al. 2006). This was attributed to the amount of nutrient delivery as well as the removal of waste and secreted factors. Although perfusion is a way to supply enough nutrients to cells for long-term culture and control the cellular microenvironment by removal of the secreted soluble factors, it disturbs the cellular diffusion-based microenvironment (Walker et al. 2004).
In this context, we developed a membrane-based two-chambered micro-bioreactor (designated as MB hereafter) and culture conditions for ESCs to investigate the influence of secreted soluble factors on cells by mimicking the diffusion-dominant in vivo microenvironment. The culture medium of the top chamber was not replaced during the culture period to avoid disturbance in the cellular microenvironment. In contrast, the culture medium of the bottom chamber was exchanged daily to maintain a sufficient nutrient supply. We cultured mESCs for five days in leukemia inhibitory factor (LIF) supplemented culture medium to study the effects of soluble factors on cellular behavior, such as cell-cell interactions, cell proliferation and differentiation, in which the influence of secreted soluble factors is important (Yu et al. 2005). In the LIF supplemented medium, BMP4 synergistically interacts with LIF to preserve the mESC pluripotency by resisting the differentiation inducing action of FGF4 (Ying et al. 2008c). Therefore, the cell states in the MB, membrane-based macro-scale Transwell Insert (TW) and conventional 6-well plate (6-WP) cultures were compared by the expression of pluripotency markers (Oct4, Sox2, Rex1 and Nanog) and cell secreted soluble factors (FGF4 and BMP4). In addition, we performed cell culture experiments by inhibiting signaling components of FGF4 and BMP4 in the MB and TW. Then, the gene expressions of inhibited and non-inhibited cultures were compared to discern the effects of soluble factors in the micro and macro-scale culture systems.
2 Materials and methods
2.1 Design of the MB
2.2 Fabrication of the MB
Details of the MB fabrication method are presented elsewhere (Kimura et al. 2008). Briefly, negative photoresist SU-8 2100 (Microchem Co.) was used to create the mold masters with the desired pattern. Then PDMS polymer (Silpot 184; Dow Corning Corp.) was mixed with its curing agent at a 10:1 ratio, poured over the mold masters, cured for 2 h at 75°C and peeled off thereafter.
The polyester membranes (pore size 0.4 μm, thickness 10 μm) were removed from Transwell Inserts 3450 (Corning Inc.). To bond the membrane with the PDMS layers, both sides of the membrane were coated with a thin layer of SiO2 by sputtering for 20 s at 150 W and 0.5 Pa. The membrane was sandwiched between the two PDMS chambers following O2 plasma treatment. Flow chips were then attached to the silicon tubing for connecting syringes; medium and reagents were manually introduced using those syringes.
2.3 Pre-treatment of experimental group (6-WP, TW and MB)
Because SiO2 was used to coat the polyester membrane incorporated in the MB, the top side of the TW membrane was also coated with SiO2 by sputtering. The TW and MB were then sterilized for 2 h under UV light. A 0.2% w/v gelatin solution was applied to cover the surface of the 6-WP and the membrane surfaces of the TW and MB, which was followed by 6 h of incubation. Culture medium for the experimental group was added to these culture systems for pre-incubation before seeding mESCs.
2.4 Routine cell culture
mESCs were routinely cultured in 60 mm gelatin coated dishes (Iwaki). Cell inoculation density was 2 × 104 cells/cm2, and the cells were passaged every other day. Culture medium composition for routine culture was high glucose DMEM (DMEM; Gibco) containing 20% ESC qualified Fetal Bovine Serum (FBS; Gibco), 1000 U/ml ESGRO-LIF (Chemicon), 1% MEM non-essential amino acids (Gibco), 2 mM GlutaMax-I (Gibco), 100 U/ml penicillin, 100 U/ml streptomycin (Gibco) and 0.1 mM 2-mercaptoethanol (Gibco). The cells were maintained in a 37°C humidified environment containing 5% CO2.
2.5 Cell culture in the experimental group
mESCs in the experimental group were cultured using the same medium as for routine culture except that DMEM and FBS were replaced with Knockout DMEM (Gibco) and 15% Knockout Serum (KSR; Gibco), respectively. KSR was used because it contains fewer extrinsic proteins. For the experimental group, cell inoculation density was 2 × 104 cells/cm2. Cell culture in the experimental group was continued for 5 days. Culture medium of the 6-WP, the lower chambers of the TW and MB were changed daily. The upper chamber culture medium of the TW and MB were not changed. Morphological examination of the cells under microscope was performed daily. For inhibition experiments, Fibroblast Growth Factor Receptor (FGFR) antagonist SU5402 (Mohammadi 1997) (Calbiochem) at 10 μM and BMP4 antagonist Noggin (Smith and Harland 1992) (R&D Systems) at 100 ng/ml were added to the culture medium.
2.6 Glucose concentration measurement
Culture medium from the 6-WP, the lower chamber of the TW and MB were collected every day during the 5-day culture. On the 5th day, the culture medium from the upper chambers of the TW and MB were also collected. Glucose concentrations were measured with a glucose analyzer (GA05, A&T Corp., Japan).
2.7 Cell collection and qPCR analysis
Genes and primers used in qPCR analyses
Cells’ self-secreted soluble factor
Cells’ self-secreted soluble factor
2.8 Statistical analysis
Student’s t-test for comparing two groups and one way ANOVA with Tukey’s post test for comparing more than two groups were performed for statistical evaluation using the demo version of GraphPad software (GraphPad Software, Inc.). Differences with a P < 0.05 (*), P < 0.01 (**), or P < 0.001 (***) were considered to be statistically significant. All data are presented as the mean ± SEM.
3.1 Effect of SiO2 coating of the membranes on ESC behavior
3.2 Cell culture condition in the 6-WP, TW and MB
3.3 Comparison of gene expression profiles among culture systems
3.4 Effects of soluble factors
We then inhibited BMP4 activity using its antagonist Noggin. Expression of the pluripotency markers Sox2 and Rex1 decreased by the Noggin treatment in the MB, but they remained unchanged in the TW (Fig. 7(b)). In addition, both in the MB and TW culture, FGF4 and BMP4 expression remained unchanged by the same treatment. Sox2 and Rex1, which were upregulated more significantly in the MB as compared to the 6-WP and TW cultures (Fig. 6), decreased significantly by the Noggin treatment (Fig. 7(b)). Therefore, we can conclude that the activity of upregulated BMP4 (Fig. 6) is responsible for the better preservation of the mESC pluripotency in the MB.
In this study, we developed a micro-scale culture system in which ESCs can be cultured in a diffusion dominant microenvironment without any limitation of nutrient supply for a long period of time. We observed better preservation of the mESC pluripotency in the micro-bioreactor than in the conventional macro-scale 6-WP and TW culture systems. We also demonstrated that autocrine effects of the up-regulated BMP4 cooperated with LIF to preserve the high pluripotency in the MB. Furthermore, the influence of FGF4 was similar in the TW and MB, whereas the influence of BMP4 was observed only in the MB.
A transcription network of Oct4, Sox2, Rex1 and Nanog maintains the pluripotency and proliferation of mESCs by suppressing the gene expression associated with differentiation (Masui et al. 2008; Niwa 2007). Usually, even in undifferentiated culture of mESCs in the presence of LIF, a proportion of the cells can undergo spontaneous differentiation (Smith 2001) which is associated with the decreased expression of those genes. Generally, overgrown differentiating mESC colonies have rough borders compared to the normal colonies. In the MB, mESC colonies were smooth-bordered, had few differentiated cells (Fig. 5(c) and (f)) and retained higher expression of the pluripotency markers (Fig. 6). These results indicated spontaneous differentiation of ESCs occurred less in the MB. Among the pluripotency markers, Sox2 and Rex1 showed prominently higher expression in the MB as compared to the WP and TW (Fig. 6). In fact, downregulation of Sox2 and Rex1 expression has a stronger correlation with loss of pluripotency of mESCs than the downregulation of Oct4 and Nanog expression (Palmqvist et al. 2005).
In spite of FGF4 accumulation, cells in the MB displayed a similar response in gene expression to that observed in the TW following FGF4 inhibition (Fig. 7(a)). However, inhibition of BMP4 activities resulted in significantly different effects in the MB and TW (Fig. 7(b)). Molecular diffusivities (inversely proportional to the cube root of molecular weight, MW) primarily determine the retention behavior of the soluble factors around the cells (Yu et al. 2005). FGF4 has a lower MW (22 kDa) than BMP4 (47 kDa), and may have diffused more quickly out of the cellular milieu. Therefore, it was unable to exert any influence on the cells as BMP4 did in the MB. In addition, extensively secreted FGF4 could have reached the threshold level of its activity equally in the MB and TW. These could be the plausible reason for the similar response observed in the TW and MB.
The average concentrations (total number of molecules divided by volume) of FGF4 and BMP4 in the MB might be the highest among the culture systems owing to the accumulation of these factors in the smallest volume. However, the cellular response to a soluble factor depends on the concentration level of the factor in the vicinity of the cell (local concentration). Both the average and local concentrations are influenced by various parameters of a soluble factor such as secretion, consumption, sequestration and release form the ECM. However, convection and diffusion only influence the local concentration. Owing to the diffusion dominant mass transfer in the MB, a soluble factor could be retained around the cells over time to reach a high concentration—all other parameters being the same in all culture systems. Therefore, in the MB, we could realize the combined effect of accumulation in a small volume and diffusion dominant mass transfer. However, we could not distinguish explicitly which concentration (average or local) reached the threshold level to impart a cellular response. To make the distinction, further investigation (experiments coupled with mathematical simulation) is necessary by taking the various parameters of a soluble factor into account along with diffusion and convection. This study, which characterizes the effects of soluble factors on ESC culture in the MB, provides a basis for the investigation.
mESCs secret FGF5, Nodal and BMP2 at low variable levels besides FGF4 and BMP4 (Wiles and Proetzel 2006). This micro-bioreactor and culture condition will be useful to study their effects in a diffusion dominant cellular environment, and will contribute to the understanding of ESC biology. The heterogeneity of ESCs during differentiation is one obstacle in obtaining lineage-specific cells useful for cell-based transplantation therapies (Singh and Terada 2007). Our micro-bioreactor can be used for obtaining relatively homogenous ESCs. In the absence of LIF, both FGF4 and BMP4 promote the differentiation of ESCs (Ying et al. 2003a). Therefore, the activity of soluble factors observed in the MB will provide an enhanced signaling microenvironment for controlling ESC differentiation process in a monolayer format such as for neuronal (Ying et al. 2003b) or hepatocyte (Teratani et al. 2005) differentiation. By keeping the cellular environment in the upper chamber minimally disturbed, it is also possible to provide other soluble factors or inhibitors through the lower chamber to gain more precise control of the differentiation process.
In this study, we developed a membrane-based two-chambered micro-bioreactor for mESC culture to mimic the diffusion dominant mass transfer environment observed in vivo. The influence of soluble factors on cells in the micro-bioreator was compared with a macro-scale culture system. We observed enhanced retention of the pluripotent phenotype of mESCs in the micro-bioreactor owing to the enhanced effect of a soluble factor in a diffusion dominant microenvironment. A similar effect of the soluble factor was not observed in the macro-scale membrane-based Transwell insert culture system, in which soluble factors dissipated away from cell surrounding through inherent convection. This micro-bioreactor offers a suitable platform not only to understand the influence of secreted soluble factors on stem cell biology, but also to address an enhanced signaling environment to direct the ESC fate.
M. M. Chowdhury was supported by Monbukagakusho scholarship from the Japan Ministry of Education, Culture, Sports, Science and Technology (MEXT). This research was supported in part by CREST from Japan Science and Technology Agency and GMSI (Global Center of Excellence for Mechanical Systems Innovation), The University of Tokyo. We would like to thank Dr. Masaki Nishikawa and Dr. Morgan Hamon for their useful suggestions regarding various technical aspects related to this study.