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Schematic drawing outlining the Steps 5–10 of the procedure to passage embryonic stem (ES) cells in suspension.
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Mouse and human embryonic stem (mES and hES) cells have become one of the most intensively studied primary cell types in biomedical research. However, culturing ES cells is notoriously labor intensive. We have optimized current ES cell culture methods by growing mES cells in suspension in a defined medium. This protocol is unsurpassed in time effic...
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... the aim to establish a cell culture platform for high- throughput mES cell research, we re-evaluated current mES culture protocols to develop a novel protocol that is simplified, rapid, robust and highly efficient (Fig. 1). We found that mES cells grown in suspension in DMEM/F12 supplemented with N2 (which contains transferrin, insulin, progesterone, putrecine and selenite 9 ), LIF and basic fibroblast growth factor (bFGF) (herein called ESN2 medium) obliviated the need for exogenous BMP and sustained mES cell cultures for extended periods. As we found ...
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The embryonic stem (ES) cell is a stem cell derived from early embryos that can indefinitely repeat self-renewing cell division cycles as an undifferentiated cell in vitro and give rise to all specialised cell types in the body. However, manipulating ES cell differentiation in vitro is a challenge due to, at least in part, heterogeneous gene induct...
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... To simplify mESC maintenance we tested protocols that replace the need for serum by a combination of two kinase inhibitors, specifically MEK inhibitor PD0325901 and GSK3 inhibitor CHIR99021 (2i) (Ying et al., 2008) and/or growth factors (BMP4) (Ying et al., 2003) and combined this with a suspension culture approach (Andang et al., 2008). ESCs cultured in serum-free 2i/LIF medium have homogeneous expression of pluripotency factors and lower lineage-associated gene expression compared to serum/LIF conditions (Marks et al., 2012;Morgani et al., 2017;Wray et al., 2010). ...
... The cell line was expanded using standard adherent culture supplemented with serum/LIF (Nichols et al., 1990;Pease et al., 1990). To establish suspension cultures we adapted a protocol developed by Andäng et al (Andang et al., 2008). The original protocol uses bFGF/LIF as self-renewal promoting factors. ...
... By modifying established suspension culture suspension protocols (Andang et al., 2008), we found that CHIR99021/LIF suspension culture condition is sufficient to maintain a homogenous pluripotent stem cell population. Most likely keeping mESC in suspension in contrast to adherent protocols significantly reduces adhesion mediated integrin signaling, which prevents mESC to differentiate (Hayashi et al., 2007). ...
In vitro stem cell culture is demanding in terms of manpower and media supplements. In recent years, new protocols have been developed to expand pluripotent embryonic stem cells in suspension culture, which greatly simplifies cell handling and scalability. However, it is still unclear how suspension culture protocols with different supplements affect pluripotency, cell homogeneity and cell differentiation compared to established adherent culture methods. Here we tested four different culture conditions for mouse embryonic stem cells (mESC) and quantified chimerism and germ line transmission as well as in vitro differentiation into three-dimensional neuro-epithelia. We found that suspension culture supplemented with CHIR99021/LIF offers the best compromise between culturing effort, robust pluripotency and cell homogeneity. Our work provides a guideline for simplifying mESC culture and should encourage more cell biology labs to use stem cell-based organoids as model systems.
... 24,25,54 Hence, the GABA function in glioma could be acquired in conjunction with the reappearance of developmental transcription factor gene programs which have been shown required for tumor propagation. 23,25,26,56 The finding of an increase of Nestin + cells and LRCs entering the cell cycle suggests that GABA activity sustains quiescence of tumor-initiating cells and when blocked, these cells rapidly re-enter the cell cycle. Because the bulk tumor produces high levels of GABA (Supplementary Figure S5), our results predict that tumor resection reduces overall GABA levels, possibly contributing to a rapid recurrence by the residual tumor cells. ...
Although genome alterations driving glioma by fueling cell malignancy have largely been resolved, less is known of the impact of tumor environment on disease progression. Here, we demonstrate functional GABAA receptor-activated currents in human glioblastoma cells and show the existence of a continuous GABA signaling within the tumor cell mass that significantly affects tumor growth and survival expectancy in mouse models. Endogenous GABA released by tumor cells, attenuates proliferation of the glioma cells with enriched expression of stem/progenitor markers and with competence to seed growth of new tumors. Our results suggest that GABA levels rapidly increase in tumors impeding further growth. Thus, shunting chloride ions by a maintained local GABAA receptor activity within glioma cells has a significant impact on tumor development by attenuating proliferation, reducing tumor growth and prolonging survival, a mechanism that may have important impact on therapy resistance and recurrence following tumor resection.Oncogene advance online publication, 4 July 2016; doi:10.1038/onc.2016.245.
... In fact, significant variation in doubling time of multiple ESC lines stemming from variation in culture conditions has previously been reported, with cell cycle transit times varying from as short as 13 h to as long as 34 h (Tamm et al. 2013). Despite the difference, ESCs grown in all culture conditions have been shown to retain their stem cell properties and could contribute to chimeras (Andang et al. 2008b;Ying et al. 2008;Smith 1991), indicating that a prolonged doubling time does not lead to loss of stem cell potential per se, and supporting our findings that pluripotency marker expression is unchanged when doubling time is increased as a response to treatment. ...
... R1/E mouse embryonic stem cell line was purchased from ATCC (lot number: 3314644) and cultured as previously described (Andang et al. 2008b). In brief, cells were seeded at 7.5 × 10 3 cells/ml in flasks for suspension culture or on laminin coated tissue culture plates for adherent culture (3 ml/well of 6-well plate, 0.5 ml/ well of 24-well plate). ...
Pluripotent stem cells are the starting cell type of choice for the development of many cell-based regenerative therapies due to their rapid and unlimited proliferation and broad differentiation potential. The unique pluripotent cell cycle underlies both these properties. Hyperpolarization-activated cyclic nucleotide-gated cation (HCN) family channels have previously been reported to modulate mouse embryonic stem cell (ESC) proliferation and here we characterize the effects of HCN inhibitor ZD7288 on ESC proliferation and stem cell identity. The doubling time of cells treated with the HCN blocker increased by ~30 % due to longer G1 and S phases, resulting in a nearly twofold reduction in ESC numbers after 4 day serum-free culture. Slower progression through S phase was not accompanied by H2AX phosphorylation or cell stalling at transition points, although EdU incorporation in treated cells was reduced. Despite the drastic cell cycle perturbations, the pluripotent status of the cells was not compromised by treatment. Cultures treated with the HCN blocker in maintenance conditions maintained pluripotency marker expression on both RNA and protein level, although we observed a reversible effect on morphology and colony formation frequency. Addition of ZD7288 in differentiating media improved FBS-driven differentiation, but not directed differentiation to neuroectoderm, further indicating that altered cell cycle structure does not necessarily compromise pluripotency and drive ESCs to differentiation. The categorically different outcomes of ZD7288 use during differentiation indicate that cell culture context can be determinative for effects of ion-modulatory molecules and underscores the need for exploring their action in serum-free conditions demanded by potential clinical use.
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The online version of this article (doi:10.1186/s40064-016-1678-7) contains supplementary material, which is available to authorized users.
... Importantly, mouse ESC proved to be a good tool since these cells can maintain the pluripotency also when are floating. A protocol published by Andang et al. 40 showed that with the presence of correct stimuli these cells keep their characteristics also when are in suspension. In addition, it has already been demonstrated that fetal microenvironment may participate in the control of ESC growth 41 , indicating that there are strong differences between adult and fetal environment. ...
Stem cell niche refers to the microenvironment where stem cells reside in living organisms. Several elements define the niche and regulate stem cell characteristics, such as stromal support cells, gap junctions, soluble factors, extracellular matrix proteins, blood vessels and neural inputs. In the last years, different studies demonstrated the presence of cKit⁺ cells in human and murine amniotic fluid, which have been defined as amniotic fluid stem (AFS) cells. Firstly, we characterized the murine cKit⁺ cells present both in the amniotic fluid and in the amnion. Secondly, to analyze the AFS cell microenvironment, we injected murine YFP⁺ embryonic stem cells (ESC) into the amniotic fluid of E13.5 wild type embryos. Four days after transplantation we found that YFP⁺ sorted cells maintained the expression of pluripotency markers and that ESC adherent to the amnion were more similar to original ESC in respect to those isolated from the amniotic fluid. Moreover, cytokines evaluation and oxygen concentration analysis revealed in this microenvironment the presence of factors that are considered key regulators in stem cell niches. This is the first indication that AFS cells reside in a microenvironment that possess specific characteristics able to maintain stemness of resident and exogenous stem cells.
... Culture media and culture conditions for ES cells have been constantly modified and improved in the past three decades [26]. Attempts have also been made to improve fES derivation efficiency in mouse strains such as 129, CBA, SCID, and some F1 hybrid strains by supplementing culture medium with small molecules. ...
The ability of small molecules to maintain self-renewal and to inhibit differentiation of pluripotent stem cells has been welldemonstrated.
Two widely used molecules are PD 98059 (PD), an inhibitor of extracellular-signal-regulated kinase 1 (ERK),
and SC1 (Pluripotin), which inhibits the RasGAP and ERK pathways. However, no studies have been conducted to compare
their effects on the pluripotency and derivation of embryonic stem (ES) cells from inbred mice C57BL/6, an important mouse
strain frequently used to model behavior, cognitive functions, immune system, and metabolic disorders in humans and also
the first mouse strain chosen to be sequenced for its entire genome. We found significantly increased derivation efficiency
of ES cells from in vivo fertilized embryos (fES) of C57BL/6 with the use of PD (71.4% over the control of 35.3%). Because fES
and ES from cloned embryos (ntES) are not distinguishable in transcription or translation profiles, we used ntES cells to
compare the effect of small molecules on their in vitro characteristics, in vitro differentiation ability, and the ability to
generate full-term ntES-4N pups by tetraploid complementation. NtES cells exhibited typical ES characteristics and upregulated
Sox2 expression in media with either small-molecule. Higher rates of full term ntES-4N pup were generated by the
supplementation of PD or SC1. We obtained the highest efficiency of ntES-4N pup generation ever reported from this strain
by supplementing ES medium with SC1. Lastly, we compared the pluripotency of fES, ntES and induced pluripotent stem
(iPS) cells of C57BL/6 background using the tetraploid complementation assay. A significant increase in implantation sites
and the number of full-term pups were obtained when fES, ntES, and iPS cells were cultured with SC1 compared to the
control ES medium. In conclusion, supplementing ES cell culture medium with PD and SC1 increases the derivation
efficiency and pluripotency, respectively, of stem cells derived from the refractory inbred C57BL/6 strain.
... R1 mouse ESCs (from A. Nagy, Toronto, Canada) were cultured as described previously (15,16). For the experiments, ESCs were grown for 48 h on tissue culture plates coated with 0.1% gelatin (Sigma) and cultured in serum-free medium (Knockout Dulbecco's modified Eagle's medium [DMEM], 15% Knockout Serum Replacement, 1× non-essential amino acids, 2 mM glutamine, 5 mM HEPES, 0.4 mM 2-mercaptoethanol [Gibco]) supplemented with 1000 U/ml ESGRO Leukemia Inhibitory Factor (Calbiochem). ...
Pluripotency of embryonic stem cells (ESCs) is maintained by transcriptional activities and chromatin modifying complexes
highly organized within the chromatin. Although much effort has been focused on identifying genome-binding sites, little is
known on their dynamic association with chromatin across cell divisions. Here, we used a modified version of the iPOND (isolation
of proteins at nascent DNA) technology to identify a large protein network enriched at nascent DNA in ESCs. This comprehensive
and unbiased proteomic characterization in ESCs reveals that, in addition to the core replication machinery, proteins relevant
for pluripotency of ESCs are present at DNA replication sites. In particular, we show that the chromatin remodeller HDAC1–NuRD
complex is enriched at nascent DNA. Interestingly, an acute block of HDAC1 in ESCs leads to increased acetylation of histone
H3 lysine 9 at nascent DNA together with a concomitant loss of methylation. Consistently, in contrast to what has been described
in tumour cell lines, these chromatin marks were found to be stable during cell cycle progression of ESCs. Our results are
therefore compatible with a rapid deacetylation-coupled methylation mechanism during the replication of DNA in ESCs that may
participate in the preservation of pluripotency of ESCs during replication.
... However, KOSR cannot alone support mES single-cell culture in the absence of feeders, and a recent study shows that, similar to FBS, it exhibits considerable lot-to-lot variability [12]. In 2008, it was shown that mES cells could be maintained in the absence of serum and feeder cells as free-floating spheres in a N2 supplemented medium with LIF and bFGF (herein named ESN2) [13,14]. In contrast to previously reported ES cell sphere cultures in media supplemented with B27 [15], the spheres grown in ESN2 do not express the neural stem cell marker nestin. ...
... In contrast to previously reported ES cell sphere cultures in media supplemented with B27 [15], the spheres grown in ESN2 do not express the neural stem cell marker nestin. However, this protocol has been reported to render mES cells prone to neurogenic differentiation [13,14]. Recently, a defined media supplemented with two inhibitors, the mitogenactivated protein kinase (MAPK)/extracellular-signal-regulated kinase (ERK) kinase (MEK) inhibitor PD0325901 and the glycogen synthase kinase 3 (GSK3) inhibitor CHIR99021, added to a B27 and N2 supplemented medium (herein named 2i) was shown to maintain mES cell self-renewal without the addition of exogenous factors [16]. ...
... Four different culture media were used in this study: 1) SCM [24], consisting of Glasgow modification of Eagles medium (GMEM) containing 5% ES-qualified fetal calf serum, 5% KnockOut TM serum replacement and 1,000 U/ml LIF (Millipore); 2) mES Prime Kit, a commercially available LIF-and FBSsupplemented complete media (PAA Laboratories/GE Healthcare); 3) 2i medium [25], a serum-free N2B27 medium supplemented with MEK inhibitor PD0325901 (1 mM) and GSK3 inhibitor CHIR99021 (3 mM) (both from Selleckchem), and 1,000 U/ml LIF (Millipore) and; 4) ESN2 [13], consisting of DMEM/F12 supplemented with N2, 10 ng/ml bFGF (R&D Systems), and 1,000 U/ml of LIF. Feeder dependent R1 [26] and C57 [27] mES cells were cultured on irradiated MEFs (R&D Systems). ...
Most stem cell laboratories still rely on old culture methods to support the expansion and maintenance of mouse embryonic stem (ES) cells. These involve growing cells on mouse embryonic fibroblast feeder cells or on gelatin in media supplemented with fetal bovine serum and leukemia inhibitory factor (LIF). However, these techniques have several drawbacks including the need for feeder-cells and/or use of undefined media containing animal derived components. Culture of stem cells under undefined conditions can induce spontaneous differentiation and reduce reproducibility of experiments. In recent years several new ES cell culture protocols, using more well-defined conditions, have been published and we have compared the standard culture protocols with two of the newly described ones: 1) growing cells in semi-adherence in a medium containing two small molecule inhibitors (CHIR99021, PD0325901) and; 2) growing cells in a spheroid suspension culture in a defined medium containing LIF and bFGF. Two feeder-dependent mouse ES (mES) cell lines and two cell lines adapted to feeder-independent growth were used in the study. The overall aim has not only been to compare self-renewal and differentiation capacity, but also ease-of-use and cost efficiency. We show that mES cells when grown adherently proliferate much faster than when grown in suspension as free-floating spheres, independent of media used. Although all the tested culture protocols could maintain sustained pluripotency after prolonged culturing, our data confirm previous reports showing that the media containing two chemical inhibitors generate more pure stem cell cultures with negligible signs of spontaneous differentiation as compared to standard mES media. Furthermore, we show that this medium effectively rescues and cleans up cultures that have started to deteriorate, as well as allow for effective adaption of feeder-dependent mES cell lines to be maintained in feeder-free conditions.
... In general, the feeder free pluripotent ES and iPS cells are difficult to maintain for extended periods as the cells tend to lose their pluripotency. Undifferentiated mouse ES cells have been maintained in bioreactors for at least 1 month [Zur Nieden et al., 2007] and in defined medium with reduced expression of integrins [Hayashi et al., 2007]; in a medium supplemented with different growth factors on serum-free systems [Andäng et al., 2008] or in feeder independent suspension cultures supplemented with polyvinyl alcohols (PVA) [Tsuji et al., 2008]. ...
... However, long-term cultures of pluripotent stem cells in suspension cultures are difficult to maintain. Short-term cultures of mouse ES cells have been tested in medium with polyvinyl alcohol (PVA) [Tsuji et al., 2008], specific growth factors [Ying et al., 2003;Andäng et al., 2008], integrin suppresses agents [Hayashi et al., 2007] as well as in bioreactors [Zur Nieden et al., 2007]. It is not been tested whether anchorage dependence is important for maintaining these cells in long-term cultures. ...
Pluripotent embryonic stem (ES) cells derived from mammalian blastocyst and the adult fibroblast derived induced pluripotent stem (iPS cells) exhibit complete potential to form cells representing all the primary germ layers such as mesoderm, endoderm and ectoderm. These cells are usually co-cultured with mouse embryonic fibroblast feeders to prevent spontaneous differentiation. Feeder free cultures can provide substantial advantage to improve the efficiency and consistency of the culture conditions. In these studies, we demonstrate that a small dietary compound retinol, the alcohol form of vitamin A has capacity to regulate the pluripotency of pluripotent stem cells and maintain highly enriched population of pluripotent ES and iPS cells in feeder free suspension cultures. Retinol maintains long-term cultures of undifferentiated cells via elevated expression of stem cell specific transcription factors Nanog and Oct4. The studies provide evidence that retinol regulates the self-renewal of pluripotent stem cells via the over expression of insulin like growth factor II (IGFII) that engages PI3 kinase signaling pathway via IGF1 receptor tyrosine kinase. The ES cells retain capacity to generate high degree germline competent chimeric animals after microinjection into blastocysts. The studies offer a convenient system for long term maintenance of pluripotent stem cells via the activation of intracellular machinery for self-renewal by a physiologically relevant compound for large-scale production of high quality pluripotent stem cells.
... These approaches risk contamination by pathogens, require separation of feeder cells from the cell type of interest, increase costs and are prone to variability. Both mouse and human embryonic stem cells (ESCs) can be maintained and expanded in a pluripotent state as floating aggregates in the absence of feeder cells [13][14][15][16] . However, all suspension cultures reported to date require serial dissociation and reaggregation steps (manipulations that typically limit cell yields). ...
... Reprogramming kinetics in adherent and suspensionculture conditions were comparable, and reprogrammed cells became independent of exogenous factor expression at the end of the processes. Commonly, suspension culture protocols for pluripotent stem cells involve repeated dissociation and reaggregation steps 14,15 . However, fibroblasts transduced with reprogramming factors were amenable to reprogramming and expansion without repeated dissociation, in the presence of doxycycline. ...
We describe derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension culture-reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor-expressing cells based on their differential survival and proliferation in suspension culture. Seamless integration of SiPSC reprogramming and directed differentiation enabled scalable production of beating cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step toward the development of robust PSC generation, expansion and differentiation technology.
... This may relate to the contribution of cell loss during passaging or culture conditions [17] and might be improved by application of stirred-suspension bioreactors [16,22,23]. These bioreactors are appealing mainly due to their simple design, scalable configuration, ease of continuous monitoring, and tight regulation of the culture environment for mouse and human ESCs [8][9][10]12,13,[37][38][39]. Such bioreactors have been used for the propagation of stem/progenitor cells including hematopoietic stem cells [40], neural precursor cells [41], and bone marrow-derived stem cells [42]. ...
Traditionally, undifferentiated pluripotent human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) have been expanded as monolayer colonies in adhesion culture, both in the presence or absence of feeder cells. However, the use of pluripotent stem cells poses the need to scale-up current culture methods. Herein, we present the cultivation of 2 hESC lines (Royan H5 and Royan H6) and 2 hiPSC lines (hiPSC1 and hiPSC4) as carrier-free suspension aggregates for an extended period of time. The cells proliferated over multiple passages kept a stable karyotype, which successfully maintained an undifferentiated state and pluripotency, as determined by marker expressions in addition to in vitro spontaneous and directed differentiation. Additionally, these cells can be easily frozen and thawed without losing their proliferation, karyotype stability, and developmental potential. Transcriptome analysis of the 3 lines revealed that the adherent culture condition was nearly identical to the suspension culture in Royan H5 and hiPSC1, but not in Royan H6. It remains unclear whether this observation at the transcript level is biologically significant. In comparison with recent reports, our study presents a low-cost procedure for long-term suspension expansion of hESCs and hiPSCs with the capability of freeze/thawing, karyotype stability, and pluripotency. Our results will pave the way for scaled up expansion and controlled differentiation of hESCs and hiPSCs needed for cell therapy, research, and industrial applications in a bioreactor culture system.
