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Culture development for human embryonic stem cell propagation: molecular aspects and challenges
Culture development for human embryonic stem cell propagation: molecular aspects and challenges BM Rao and PW Zandstra Basic fibroblast growth factor and members of the transforming growth factor-b superfamily are important regulators of human embryonic stem cell (hESC) self-renewal. Extensive cross-talk between the intracellular signaling pathways activated by these factors contributes to maintenance of the undifferentiated hESC state. Understanding the molecular regulation of hESC self-renewal will facilitate the design of improved systems for hESC propagation and provide a foundation for strategies to direct the differentiation of hESCs to clinically relevant cell types. Addresses Institute of Biomaterials and Biomedical Engineering, Room 407, Roseburgh Building, 4 Taddle Creek Road, Toronto, Ontario, M5S 3G9, Canada Corresponding author: Zandstra, PW ([email protected])
Current Opinion in Biotechnology 2005, 16:568–576 This review comes from a themed issue on Biochemical engineering Edited by Govind Rao Available online 11th August 2005 0958-1669/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2005.08.001
Introduction Human embryonic stem cells (hESCs) were isolated from the inner cell mass of blastocyst-stage embryos [1]. These cells can be propagated in culture for extended periods and can differentiate into all somatic cell types, underlying their great potential as a renewable source of functional cells for therapeutic applications. To realize this potential, however, it is necessary to devise robust and cost-effective systems for culturing undifferentiated hESCs at clinically relevant scales, and to develop strategies to direct the differentiation of hESCs to specific functional cell types. In this review we discuss issues specifically related to the development of robust systems for hESC propagation. We present advances in the development of culture systems for the maintenance of undifferentiated hESCs, discuss the current understanding of the molecular regulation of hESC selfrenewal, and identify key challenges that need to be addressed to facilitate the development of hESC-based therapeutic applications. Current Opinion in Biotechnology 2005, 16:568–576
Current hESC culture systems hESCs were originally derived in medium containing serum, with the support of feeder layers of mouse embryonic fibroblasts (mEFs) that secrete factors essential for maintaining hESCs in the undifferentiated state [1]. However, serum is a complex mixture of proteins of unknown composition, and different batches of serum exhibit variability. A defined culture system with feederfree conditions and medium of known protein composition is desirable to minimize the variability in culture conditions that affects the reproducibility and robustness of hESC culture. From the perspective of therapeutic applications, it might also be important for hESCs to be cultured (and derived) under xeno-free conditions (i.e. conditions free of components derived from animal sources). Indeed, hESCs cultured on feeders with medium containing serum components derived from animal sources express, at least transiently, an immunogenic nonhuman sialic acid and might not be immediately suitable for therapeutic applications [2]. The first feeder-layer-free cultures of hESCs used medium conditioned by the factors secreted by mEFs and MatrigelTM- or laminin-coated plastic plates [3]. MatrigelTM is a complex mixture of mouse sarcoma origin and contains mostly laminin, collagen IV, heparan sulfate proteoglycans (HSPGs) and entactin. Animal serum was replaced by a more defined serum replacement formulation called KnockOut Serum Replacement (SR) that contains components such as insulin, transferrin and lipid-rich bovine albumin [4]. Subsequently, the use of medium (containing SR) conditioned by hESC-derived fibroblasts (hEFs) immortalized by ectopic overexpression of telomerase to maintain hESCs in an undifferentiated state, was also reported [5]. This finding illustrates an important aspect of hESC culture that has to be considered during culture development — differentiated cells generated in non-supportive or poorly supportive hESC cultures might produce factors that promote or prolong hESC maintenance. Amit et al. [6] reported the maintenance of hESCs on a fibronectin matrix using unconditioned medium (i.e. medium containing SR but not conditioned by mEFs or hEFs) supplemented with basic fibroblast growth factor (bFGF), also known as fibroblast growth factor 2 (FGF2), and transforming growth factor-b1 (TGF-b1). However, hESCs cultured under these conditions have lower cloning efficiencies and growth rates and higher rates of differentiation than cells grown on mEF-conditioned media. HESCs have also been maintained in the undifferentiated state on MatrigelTM-coated plates in unconditioned medium www.sciencedirect.com
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Table 1 Feeder-free culture of human embryonic stem cells. Cell adhesion factor TM
Medium composition
Comments
Matrigel or laminin Human or bovine fibronectin
80% KO-DMEM; 20% KO-SR; mEF-CM + 4ng/mL bFGF 85% KO-DMEM; 15% KO-SR; 0.12 ng/mL TGF-b1; 4 ng/mL bFGF
MatrigelTM
80% KO-DMEM; 20% KO-SR; 24–36 ng/mL bFGF
MatrigelTM MatrigelTM
80% KO-DMEM; 20% KO-SR; 40 ng/mL bFGF 80% KO-DMEM; 20% KO-SR Medium conditioned by hES-derived fibroblasts, immortalized by ectopic overexpression of telomerase 80% DMEM/F12; 20% KO-SR; 500 ng/mL noggin; 40 ng/mL bFGF 80% KO-DMEM; 20% KO-SR; 50 ng/mL activin A; 50 ng/mL KGF; 10 mM nicotinamide
MatrigelTM Laminin
Lower cloning efficiency and growth rates and higher differentiation rates than cells grown on mEFs Different batches of bFGF required different working concentrations
Ref. [3] [6]
[8] [7] [5]
Higher concentrations of bFGF (100 ng/mL) can replace noggin
[9,10] [11]
In all cases, cell culture medium is supplemented with 0.1 mM b-mercaptoethanol, 1% non-essential amino acids stock and 1 mM (except in [6] where 2 mM L-glutamine was used). DMEM, Dulbecco’s modified Eagle medium; mEF-CM, SR-containing medium conditioned by mEFs.
L-glutamine
supplemented with high concentrations of bFGF [7,8], a combination of the bone morphogenic protein (BMP) antagonist noggin and bFGF [9,10] or a combination of keratinocyte growth factor (KGF, a member of the FGF family), nicotinamide and activin A [11]. A list of different culture systems reported for the maintenance of undifferentiated hESCs is presented in Table 1, highlighting the significant advances that have been made towards developing a defined culture system. However, components from animal sources have not been completely eliminated; SR and MatrigelTM both contain complex components of non-human origin. The use of human laminin to replace MatrigelTM could solve the problem in part, although significant variability exists in both human and mouse laminin obtained from different manufacturers and in different lots from the same manufacturer [9]. An interim solution to the problem of eliminating animal-derived components in hESC culture is the use of human feeders and human serum. Richards et al. [12] have demonstrated the derivation and propagation of a hESC line in completely xeno-free conditions. Table 2
summarizes the various sources of human feeders that have been validated for culture of undifferentiated hESCs [13–18]. Although a great deal of progress has been made in establishing better-defined conditions for the culture of hESCs, as is evident in Tables 1 and 2, the ultimate goal of a robust feeder-free system with a completely defined culture medium, where all components used are derived entirely from non-animal sources, remains elusive (see Update).
Extracellular factors regulating hESC self-renewal Work cited in the previous section indicates that the undifferentiated hESC state can be maintained by extracellular cues provided by the extracellular matrix (ECM) proteins, such as laminin, and exogenously added factors of the TGF-b and FGF families. The role of laminins in development has been discussed elsewhere [19]. Here we specifically examine the role of members of the TGF-b and FGF families in the regulation of hESC self-renewal.
Table 2 Culture of human embryonic stem cells on feeders from human sources. Feeder source
Comment
Ref.
Fetal muscle, fetal skin, adult fallopian tube Neonatal foreskin Adult skin, adult muscle Marrow stromal cells Adult uterine endometrial cells, adult breast parenchymal cells, embryonic fibroblasts Human embryonic stem cell line
Proof of derivation and propagation of a hESC line in completely xeno-free conditions
[12]
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A comparison of hES culture using human feeders from different sources hESCs grown on human uterine endometrial cells and embryonic fibroblasts have slightly different morphology (thinner and flatter) than cells grown on mEF An autogeneic feeder cell system is described. hESCs can also be cultured in feeder-free conditions using medium conditioned by autogeneic feeders.
[13,14] [15] [16] [17]
[18]
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The transforming growth factor-b superfamily
Signaling mediated by the ligands of the TGF-b superfamily has been previously reviewed by Shi and Massague [20]. Briefly, signaling is initiated by ligand binding to type I and type II receptors that have serine/threonine kinase activity. Ligand binding leads to phosphorylation of the type I receptor by the type II receptor and subsequent phosphorylation of Smad (mothers against decapentaplegic homolog [drosophila]) proteins by the type I receptor. The TGF-b superfamily ligands can be classified into two subfamilies: the TGF-b/activin/nodal subfamily that signals through the type I receptors activin receptor-like kinases (ALKs) ALK2, ALK4, ALK5, ALK7 and the receptor Smads (R-Smads) Smad2 and Smad3; and the BMP/growth and differentiation factor (GDF) subfamily that signals through the type I receptors ALK1, ALK3, ALK6 and the R-Smads Smad1, Smad5 and Smad8. Upon phosphorylation by the type I receptor tyrosine kinase, the R-Smads associate with a co-mediator Smad (C-Smad) Smad4 and translocate to the nucleus. The activated Smad complexes in the nucleus regulate the transcription of several target genes, in conjunction with other cofactors. Two inhibitory Smads (I-Smads), Smad6 and Smad7, negatively regulate signaling through the R-Smads. Smad7 inhibits all R-Smads, whereas Smad6 selectively inhibits Smad1. Gene expression analysis has shown that the genes associated with TGF-b and BMP signaling are expressed in hESCs [21,22]. The role of the BMP/GDF subfamily
Xu et al. [23] and Pera et al. [24] have reported two different effects of the BMP/GDF subfamily on hESCs. In the hESC line H1 cultured on MatrigelTM-coated plates with conditioned medium from mEFs, Xu and colleagues report differentiation of hESCs along the trophoblast lineage in response to BMP-4. A similar effect is observed in response to BMP-2, BMP-7 and GDF-5. In the case of hESC lines HES2 and HES3 cultured on feeders, the addition of BMP-2, BMP-4 or BMP-7 leads to differentiation to primitive endoderm [24]. Nevertheless, the undifferentiated hESC state is characterized by low levels of phosphorylated Smad1/5/8 [9,24,25], suggesting that suppression of BMP mediated signaling (i.e. inhibition of Smad1/5/8) is necessary for hESC maintenance. Indeed, medium conditioned by mEFs used in the maintenance of hESC cultures contains the BMP antagonists noggin and gremlin [9]. Also, hESCs can be maintained in the undifferentiated state in feeder-free conditions using unconditioned medium (medium containing SR but not conditioned by mEFs) supplemented with bFGF and noggin [9,10]. Some level of BMP signaling, however, might be important for maintenance of the undifferentiated hESC state. When noggin is added to hESC cultures on feeders, cells with neuroectodermal markers are obtained indicating that BMPmediated signaling might prevent differentiation to Current Opinion in Biotechnology 2005, 16:568–576
neuroectoderm [24]. This is consistent with data obtained using mouse embryonic stem cells where BMP-4 inhibits induction of neuroectodermal differentiation [26]. It is interesting to note the different effects of BMP on hESCs cultured on feeders (differentiation to primitive endoderm) from those cultured in feeder-free conditions with unconditioned medium (differentiation to trophoblast). One possible explanation for this difference, besides variability between cell lines used, is that the feeder layers provide other factors that inhibit BMP signaling. For instance, medium conditioned by mEFs contains activin A, which suppresses BMP signaling (elaborated further in the following section). Thus, the undifferentiated hESC state can be regulated in part through a balance between BMP signaling to inhibit neuroectodermal differentiation and the activity of BMP antagonists, such as noggin, involved in blocking BMP-induced differentiation to primitive endoderm and/ or trophoblast. The role of the TGF-b/activin/nodal subfamily
The undifferentiated hESC state is characterized by high levels of phosphorylated Smad2/3, indicating an important role for signaling mediated by the TGF-b/activin/ nodal subfamily in the regulation of hESC self-renewal [9,25,27]. SR-containing unconditioned medium exhibits BMP-like differentiation activity (either directly as a component of SR or indirectly through factors in SR that induce autocrine secretion of BMP) as seen by high levels of phosphorylated Smad1/5/8, detectable after 24 h culture with hESC, along with BMP-2 and BMP-4 proteins [9]. TGF-b1 [6] or activin A [11], together with bFGF, seem able to maintain undifferentiated hESCs under feeder-free conditions in SR-containing unconditioned medium. By themselves, in the same medium, TGF-b [6] or activin A [11] maintain the undifferentiated state, but cell proliferation is reduced. Also, medium conditioned by mEFs contains activin A [11]. Thus, the TGF-b/activin/nodal subfamily appears to suppress BMP signaling through Smad1/5/8. As discussed earlier, because BMP signaling drives differentiation to trophoblast and/or primitive endoderm, the TGF-b/activin/ nodal subfamily presumably suppresses differentiation to these lineages. Signaling through Smad2/3 leads to increased expression of nodal and the nodal antagonists lefty-A and lefty-B [27]. Nodal is known to prevent differentiation of hESCs along the neuroectodermal pathway [28]. Downregulation of nodal, lefty-A and lefty-B gene expression is one of the earliest events upon differentiation induced by withdrawal of conditioned medium [27]. Nodal can bind BMP in the extracellular milieu, leading to inhibition of the effects mediated by both nodal and BMP [29]. Thus, Smad2/3 signaling negatively regulates signaling through Smad1/5/8 and the effect of nodal (and hence Smad2/3) through expression of the lefty proteins. www.sciencedirect.com
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The fibroblast growth factor family
The other major player in regulating hESC self-renewal is the fibroblast growth factor (FGF) family, extensively reviewed in [30–32]. The FGF family consists of 22 ligands signaling through four cell-surface FGF receptors (FGFR1–4) with intrinsic tyrosine kinase activity. Alternative splicing leads to numerous isoforms of FGFR. Each FGFR can bind to multiple FGF ligands. In addition, HSPGs act as low-affinity receptors for the FGF ligands and stabilize the formation of the FGF–FGFR complex. FGF binding to FGFR induces receptor dimerization and initiates tyrosine kinase activity and subsequent activation of the Ras-mitogen associated protein kinase (MAPK), phosphatidylinositol-3 (PI3) kinase and phospholipase C-g (PLC-g) pathways. Gene expression analysis indicates that FGF signaling is active in undifferentiated hESCs [21,22] (see Update). Indeed, bFGF is an important exogenously added factor in the medium used to culture undifferentiated hESCs. In the absence of bFGF, the undifferentiated hESC state can be maintained in unconditioned medium by TGF-b1 or activin but the cells proliferate poorly [6], suggesting that bFGF plays a role in hESC mitogenesis [33]. Also, signaling through the PI3 kinase/Akt/protein kinase B (PKB) pathway initiated by bFGF has been implicated in the expression of ECM molecules [34]. High concentrations of bFGF can replace noggin in maintaining the undifferentiated hESC state in unconditioned medium, thus suppressing the BMP-like differentiation activity induced by unconditioned medium [9]. However, under these conditions, phosphorylation of Smad1 is still observed. A likely explanation for this phenomenon is that MAPK activated by bFGF signaling prevents nuclear localization of phosphorylated Smad1 (explained in the following section) [35].
Interactions regulating the undifferentiated hESC state Self-renewal of hESCs is regulated by exogenously added and autocrine factors the interact with each other and the cells in the extracellular milieu. Interactions between these factors in an extracellular context and the crosstalk between the intracellular signaling pathways activated by them contribute to maintenance of the undifferentiated hESC state. Figure 1a illustrates the balance between different known factors in the extracellular milieu that maintain the hESC in the undifferentiated state; Table 3 is a concise compilation of the information used to construct Figure 1a. Important aspects of the crosstalk between the signaling pathways activated by these extracellular factors is shown in Figure 1b. Outside the cell, binding interactions between BMP and noggin [9,10] and BMP and nodal [29] negatively regulate BMP signaling. Binding between BMP and nodal also inhibits nodal signaling [29]. The nodal antagonists lefty-A and lefty-B inhibit nodal signaling by www.sciencedirect.com
directly interacting with nodal, thereby preventing binding to its receptor complex or interacting with the EGF-CFC proteins (accessory receptors for nodal) and preventing formation of the nodal receptor complex [36]. HSPGs present on the cells as well as in the ECM bind to FGF and can regulate FGF signaling. Intracellularly, significant cross-talk exists between the FGF and TGF-b/BMP signaling pathways. The R-Smads contain an N-terminal MH1 domain and a C-terminal MH2 domain. Phosphorylation of the C-terminal MH2 domain by the type I receptor kinase is necessary for activation of the R-Smads [20]. The linker region between the MH1 and MH2 domains also contain phosphorylation sites that mediate crosstalk between the Smad and MAPK signaling pathways. Phosphorylation of the linker region of R-Smads by MAPK alters the nuclear localization and hence activity of the R-Smads. Linker phosphorylation of Smad1 and Smad2 prevents nuclear localization, whereas linker-phosphorylated Smad3 is localized in the nucleus [35,37]. Thus, the biological activity of the R-Smads is regulated by MAPK signaling. Members of the TGF-b superfamily, bFGF and the lefty proteins can all activate MAPK signaling [20,30,32,38]. The phosphorylation of Smad3 at the C-terminal MH2 domain is inhibited by direct interaction with Akt/PKB [39,40]. As discussed earlier, FGF mediates signaling through the PI3 kinase/Akt/PKB pathway. Gene expression analysis suggests that the Wnt signaling pathway might be active in undifferentiated hESCs [22], although the role of Wnt in hESC maintenance is unclear. The hESC undifferentiated state and corresponding high levels of phosphorylated Smad2/3 can be maintained in unconditioned medium, at least over short periods of time, in the presence of a glycogen synthase kinase 3-b (GSK3-b) inhibitor [25,41]. The level of phosphorylated Smad2/3 decreases in the presence of an inhibitor that prevents phosphorylation of Smad2/3 by the type I TGF-b receptors, suggesting that the inhibition of GSK3b is associated with expression of factors that act in the extracellular milieu and are involved in Smad2/3 signaling [25]. Cross-talk also exists between the FGF and Wnt signaling pathways [30]. Akt/PKB phosphorylates GSK3b leading to inhibition of GSK3-b activity. It is interesting to note that the Wnt, FGF and TGF-b signaling pathways converge at GSK3-b, thereby implicating it as a convergence node in these interacting signaling networks. Together, these signaling results support an emerging perspective that hESCs, like mESCs [26] (and perhaps stem cells in general), maintain their undifferentiated state by inhibiting signaling (and gene expression) programs that lead to the development of specific cell types. At the signaling level, interactions between extracellular factors and crosstalk between signaling pathways may be an important part of this negative regulation. It is Current Opinion in Biotechnology 2005, 16:568–576
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Figure 1
Molecular interactions regulating hESC self-renewal. (a) Extracellular factors regulating hESC self-renewal and their interactions. Red lines indicate negative regulation; green arrows show positive regulation. Solid lines denote extracellular interactions; dashed lines indicate intracellular interactions or indirect effects. (b) Cross-talk between intracellular signaling pathways regulating hESC self-renewal. Red lines indicate negative regulation; green arrows show positive regulation. Solid lines denote direct interactions; dashed lines indicate indirect effects.
particularly appealing, therefore, to consider the ESC as an unstable steady state [42] (as shown in Figure 2) balanced in a high energy local minimum in the energy landscape, with a tendency upon perturbation to differentiate (to a lower energy state) along different lineages. Understanding this balance, as well as how to systematically perturb it, will be an important aspect of hESC research in the next few years.
Near-term challenges in hESC culture development Recent advances in stem cell biology have led to a clearer definition of the players involved in hESC self-renewal and the concomitant development of better-defined conditions for undifferentiated hESC culture. However, current understanding is far from perfect and several key Current Opinion in Biotechnology 2005, 16:568–576
challenges need to be overcome for the culture of undifferentiated hESCs in a therapeutically relevant manner. A major problem in the design of bioprocesses for hESC propagation is the need for the development of rapid, robust and predictive assays for screening the impact of culture manipulations on hESC maintenance. The classic definition of the ESC state is largely based on the mESC paradigm. Simplistically, an ESC (as defined in the context of the mESC) can be propagated extensively in culture, has a normal diploid karyotype, and can give rise to cells of all embryo-derived lineages in vivo and in vitro. In hESCs, the ability of the cells to recapitulate all aspects of in vivo embryogenesis cannot be validated. Currently a hESC culture system is considered supportive if the cells retain cell surface and intracellular markers associated www.sciencedirect.com
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Table 3 Effect of different factors on the regulation of human embryonic stem cell self-renewal. Factor
Effects
Ref.
BMP
BMP promotes differentiation to trophoblast BMP promotes differentiation to primitive endoderm Noggin promotes self-renewal by antagonizing BMP Presence of noggin leads to the upregulation of neuronal markers Therefore, BMP prevents differentiation to neuroectoderm
[23] [24] [7,10] [24] (See update)
Nodal
Nodal prevents differentiation to neuroectoderm Activation of Smad2/3 by nodal leads to expression of nodal and the nodal antagonists lefty-A and lefty-B Nodal binds to and inhibits BMP
[28] [27]
Activin A
Activin A can maintain hESC undifferentiated state in the presence of FGF Inhibition of BMP signaling is necessary to maintain hESC undifferentiated state Therefore, activin A inhibits the effect of BMP signaling
[11] [9,10]
TGF-b
TGF-b can maintain undifferentiated hESC state in the presence of FGF Inhibition of BMP signaling is necessary to maintain hESC cell undifferentiated state Therefore, TGF-b inhibits the effect of BMP signalling
[6,11] [9,10]
bFGF
High concentrations of bFGF can replace noggin to maintain the undifferentiated hESC state Therefore, BMP signaling is antagonized by bFGF
[9]
[29]
Statements in italics are inferences deduced from the references cited.
with (and previously defined to be correlated with) hESC pluripotency, are karyotypically normal, and are able to differentiate into (functional) cells of ectoderm, mesoderm and endoderm lineages — a property typically detected in vitro using embryoid body formation assays and/or in vivo using teratoma formation assays in mice (reviewed in [43]). Thus, the currently accepted paradigm for assaying the pluripotency of hESCs involves tedious and lengthy experimental procedures and does not typically allow for the interrogation of hESC properties at the clonal level (a criterion necessary to rigorously establish quantitative relationships between the microenvironment and stem cell maintenance). A rapid assay that uses quantifiable parameters such as concentration or the behavior of particular molecular species and reliably predicts individual hESC developmental potential is highly desirable. Several studies report the use of gene
expression analysis to identify a set of genes that are differentially expressed in pluripotent hESCs, and hence assign a unique molecular signature to undifferentiated hESCs [44,45] (see Update). In the mESC system, Palmqvist et al. [46] reported the correlation of gene expression profiles using functional assays that assess developmental potential to identify genes that are downregulated during early differentiation. However, in the case of hESCs, similar studies that compare gene expression analysis to functional measures of pluripotency (hence validating a putatively unique signature) have not been reported. An alternate approach might be to use hESC signaling responsiveness to define a unique molecular signature for undifferentiated hESCs. Understanding unique aspects of static or dynamic hESC signaling networks will be important if this approach is to prove fruitful.
Figure 2
The hESC modeled as an unstable steady state. Different factors maintain the steady state by preventing differentiation along different paths (e.g. noggin and bFGF prevent differentiation to the trophoblast by blocking BMP signaling). www.sciencedirect.com
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As discussed earlier, several media formulations have been used for culturing undifferentiated hESCs; however, hESC culture systems predominantly use MatrigelTM as the ECM substrate for cell attachment. The development of engineered attachment substrates for the culture of hESCs remains an important challenge. Understanding the interactions between signaling pathways initiated by the cytokines in the medium and signaling associated with cell adhesion and spreading on the ECM will facilitate the design of improved attachment substrates [47]. Anderson et al. [48] have developed a highthroughput system for synthesizing and evaluating novel substrates for surface attachment of hESCs. The use of such high-throughput platforms (and those described elsewhere [49]) in conjunction with predictive screening-based assays for hESC developmental potential could be an effective approach for hESC culture development. Finally, defined and robust culture systems will have to be integrated with cost-effective strategies for the generation of significant numbers of validated hESCs under good manufacturing practice (GMP) conditions. Microenvironmental parameters such as dissolved oxygen, pH and media supplementation play an important role in ESC culture (see Update). For instance, dissolved oxygen affects both ESC self-renewal and differentiation; low oxygen tension has been shown to prevent the differentiation of hESCs [50]. In the mESC system, hypoxic conditions improve the yield of ESC-derived cardiomyoctes [51]. Low levels of oxygen are likely to affect ESC signaling pathways through the hypoxia-response element inducible proteins. In mESCs, survival under prolonged hypoxic conditions (48 h) is mediated by autocrine vascular endothelial growth factor, which is upregulated by hypoxia-inducible transcription factors [52]. A systematic analysis of the impact of the dynamic interactions between culture variables such as oxygen tension and intracellular signaling that controls ES cell fate will be important for the development of robust stem-cell technologies. The development of bioreactors based on an in-depth understanding of controllable, seemingly pedestrian but nonetheless critical, microenvironmental parameters will be required for the generation of therapeutically useful hESCs.
specific differentiated cell types from hESCs, a multistep process that might involve tipping the balance between contrasting cell fates at multiple steps along the way.
Update A demonstration of a defined and xeno-free culture system for undifferentiated hESCs has recently been reported by Li et al. [53]. hESCs were cultured on surfaces coated with laminin of human placental origin, using a commercially available defined serum-free medium that does not contain animal-sourced products, supplemented with high concentrations of human bFGF. The role of bFGF in hESC culture has been further elaborated in an analysis that suggests a direct influence of signaling mediated by endogenous bFGF on hESC self-renewal [54]. In another recent study, Yang et al. [55] have reported the development of a focused microarray that will assist in evaluating the effect of culture microenvironment on hESC self-renewal and differentiation. Gerrard et al. [56] report hESC differentiation to neural lineages, in attached cultures, upon blocking of BMP signaling by noggin. The importance of cellular microenvironment in hESC culture is further demonstrated in a recent study by Saha et al. [57], which shows that the mechanical agitation of the cell substratum results in the inhibition of hESC differentiation relative to unmanipulated controls.
Acknowledgements We apologize to our colleagues whose work was not cited in this review. Thanks to Ryan Davey and Jonathan Draper for helpful comments. BMR is supported by the Canadian Stem Cell Network and the Stem Cell Biology and Regenerative Medicine Center at the Robarts Research Institute (London, Ontario). PWZ is the Canada Research Chair in Stem Cell Bioengineering.
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Conclusions Understanding the molecular mechanisms governing hESC self-renewal will facilitate the design of robust assays for hESC pluripotency, which in turn will drive the development of improved systems for culturing undifferentiated hESCs. Defined culture systems will greatly help in the generation of quantitative models for the molecular regulation of self-renewal versus early differentiation decisions. A predictive systems-level understanding of the molecular mechanisms involved in the regulation of hESC fate underlies the development of rational strategies for obtaining pure populations of Current Opinion in Biotechnology 2005, 16:568–576
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12. Richards M, Fong CY, Chan WK, Wong PC, Bongso A: Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol 2002, 20:933-936. Proof of derivation and propagation of a hESC line in completely xenofree conditions is demonstrated.
27. Besser D: Expression of nodal, lefty-A, and lefty-B in undifferentiated human embryonic stem cells requires activation of Smad2/3. J Biol Chem 2004, 279:45076-45084. Nodal and nodal antagonists lefty-A and lefty-B are downregulated very early upon initiation of hESC differentiation. Expression of lefty and nodal is regulated by Smad2/3 in hESCs.
13. Amit M, Margulets V, Segev H, Shariki K, Laevsky I, Coleman R, Itskovitz-Eldor J: Human feeder layers for human embryonic stem cells. Biol Reprod 2003, 68:2150-2156.
28. Vallier L, Reynolds D, Pedersen RA: Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev Biol 2004, 275:403-421.
14. Hovatta O, Mikkola M, Gertow K, Stromberg AM, Inzunza J, Hreinsson J, Rozell B, Blennow E, Andang M, Ahrlund-Richter L: A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod 2003, 18:1404-1409.
29. Yeo C, Whitman M: Nodal signals to Smads through Criptodependent and Cripto-independent mechanisms. Mol Cell 2001, 7:949-957.
15. Richards M, Tan S, Fong CY, Biswas A, Chan WK, Bongso A: Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. Stem Cells 2003, 21:546-556. 16. Cheng L, Hammond H, Ye Z, Zhan X, Dravid G: Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells 2003, 21:131-142. 17. Lee JB, Song JM, Lee JE, Park JH, Kim SJ, Kang SM, Kwon JN, Kim MK, Roh SI, Yoon HS: Available human feeder cells for the maintenance of human embryonic stem cells. Reproduction 2004, 128:727-735. 18. Stojkovic P, Lako M, Stewart R, Przyborski S, Armstrong L, Evans J, Murdoch A, Strachan T, Stojkovic M: An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 2005, 23:306-314. 19. Li S, Edgar D, Fassler R, Wadsworth W, Yurchenco PD: The role of laminin in embryonic cell polarization and tissue organization. Dev Cell 2003, 4:613-624. 20. Shi Y, Massague J: Mechanisms of TGF-b signaling from cell membrane to the nucleus. Cell 2003, 113:685-700. 21. Brandenberger R, Wei H, Zhang S, Lei S, Murage J, Fisk GJ, Li Y, Xu C, Fang R, Guegler K et al.: Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat Biotechnol 2004, 22:707-716. 22. Wei CL, Miura T, Robson P, Lim SK, Xu XQ, Lee MY, Gupta S, Stanton L, Luo Y, Schmitt J et al.: Transcriptome profiling of human and murine ESCs identifies divergent paths required to maintain the stem cell state. Stem Cells 2005, 23:166-185. www.sciencedirect.com
30. Dailey L, Ambrosetti D, Mansukhani A, Basilico C: Mechanisms underlying differential responses to FGF signaling. Cytokine Growth Factor Rev 2005, 16:233-247. 31. Mohammadi M, Olsen SK, Ibrahimi OA: Structural basis for fibroblast growth factor receptor activation. Cytokine Growth Factor Rev 2005, 16:107-137. 32. Eswarakumar VP, Lax I, Schlessinger J: Cellular signaling by fibroblast growth factor receptors. Cytokine Growth Factor Rev 2005, 16:139-149. 33. Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA: Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 2000, 227:271-278. 34. Kim SJ, Cheon SH, Yoo SJ, Kwon J, Park JH, Kim CG, Rhee K, You S, Lee JY, Roh SI et al.: Contribution of the PI3K/Akt/PKB signal pathway to maintenance of self-renewal in human embryonic stem cells. FEBS Lett 2005, 579:534-540. 35. De Robertis EM, Kuroda H: Dorsal-ventral patterning and neural induction in Xenopus embryos. Annu Rev Cell Dev Biol 2004, 20:285-308. 36. Chen C, Shen MM: Two modes by which Lefty proteins inhibit nodal signaling. Curr Biol 2004, 14:618-624. 37. Yamagata H, Matsuzaki K, Mori S, Yoshida K, Tahashi Y, Furukawa F, Sekimoto G, Watanabe T, Uemura Y, Sakaida N et al.: Acceleration of Smad2 and Smad3 phosphorylation via c-Jun NH2-terminal kinase during human colorectal carcinogenesis. Cancer Res 2005, 65:157-165. 38. Ulloa L, Creemers JW, Roy S, Liu S, Mason J, Tabibzadeh S: Lefty proteins exhibit unique processing and activate the MAPK pathway. J Biol Chem 2001, 276:21387-21396. Current Opinion in Biotechnology 2005, 16:568–576
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39. Conery AR, Cao Y, Thompson EA, Townsend CM Jr, Ko TC, Luo K: Akt interacts directly with Smad3 to regulate the sensitivity to TGF-b induced apoptosis. Nat Cell Biol 2004, 6:366-372. 40. Remy I, Montmarquette A, Michnick SW: PKB/Akt modulates TGF-b signalling through a direct interaction with Smad3. Nat Cell Biol 2004, 6:358-365. 41. Sato N, Meijer L, Skaltsounis L, Greengard P, Brivanlou AH: Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor. Nat Med 2004, 10:55-63. 42. Waddington CH, Robertson E: Selection for developmental canalisation. Genet Res 1966, 7:303-312. 43. Hoffman LM, Carpenter MK: Characterization and culture of human embryonic stem cells. Nat Biotechnol 2005, 23:699-708. 44. Sato N, Sanjuan IM, Heke M, Uchida M, Naef F, Brivanlou AH: Molecular signature of human embryonic stem cells and its comparison with the mouse. Dev Biol 2003, 260:404-413. 45. Bhattacharya B, Miura T, Brandenberger R, Mejido J, Luo Y, Yang AX, Joshi BH, Ginis I, Thies RS, Amit M et al.: Gene expression in human embryonic stem cell lines: unique molecular signature. Blood 2004, 103:2956-2964. 46. Palmqvist L, Glover CH, Hsu L, Lu M, Bossen B, Piret JM, Humphries RK, Helgason CD: Correlation of murine embryonic stem cell gene expression profiles with functional measures of pluripotency. Stem Cells 2005, 23:663-680. 47. Prudhomme W, Daley GQ, Zandstra P, Lauffenburger DA: Multivariate proteomic analysis of murine embryonic stem cell self-renewal versus differentiation signalling. Proc Natl Acad Sci USA 2004, 101:2900-2905. 48. Anderson DG, Levenberg S, Langer R: Nanoliter-scale synthesis of arrayed biomaterials and application to human embryonic stem cells. Nat Biotechnol 2004, 22:863-866.
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Describes a platform for the high-throughput synthesis and evaluation of novel substrates that mediate cell attachment. 49. Flaim CJ, Chien S, Bhatia SN: An extracellular matrix microarray for probing cellular differentiation. Nat Methods 2005, 2:119-125. 50. Ezashi T, Das P, Roberts RM: Low O2 tensions and the prevention of differentiation of hES cells. Proc Natl Acad Sci USA 2005, 102:4783-4788. The suppressive effect of low oxygen tension on differentiation of hESCs is described. 51. Bauwens C, Yin T, Dang S, Peerani R, Zandstra PW: Development of a perfusion fed bioreactor for embryonic stem cell-derived cardiomyocyte generation: oxygen-mediated enhancement of cardiomyocyte output. Biotechnol Bioeng 2005, 90:452-461. 52. Brusselmans K, Bono F, Collen D, Herbert JM, Carmeliet P, Dewerchin M: A novel role for vascular endothelial growth factor as an autocrine survival factor for embryonic stem cells during hypoxia. J Biol Chem 2005, 280:3493-3499. 53. Li Y, Powell S, Brunette E, Lebkowski J, Mandalam R: Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products. Biotechnol Bioeng 2005: in press. 54. Dvorak P, Dvorakova D, Koskova S, Vodinska M, Najvirtova M, Krekac D, Hampl A: Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells. Stem Cells 2005: in press. 55. Yang AX, Mejido J, Luo Y, Zeng X, Schwartz C, Wu T, Thies RS, Bhattacharya B, Han J, Freed B et al.: Development of a focused microarray to assess human embryonic stem cell differentiation. Stem Cells Dev 2005, 14:270-284. 56. Gerrard L, Rodgers L, Cui W: Differentiation of human embryonic stem cells to neural lineages in adherent culture by blocking BMP signaling. Stem Cells 2005: in press. 57. Saha S, Ji L, de Pablo JJ, Palecek SP: Inhibition of human embryonic stem cell differentiation by mechanical strain. J Cell Physiol 2005: in press.
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Current Opinion in Biotechnology 2005, 16:568–576 This review comes from a themed issue on Biochemical engineering Edited by Govind Rao Available online 11th August 2005 0958-1669/$ – see front matter # 2005 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2005.08.001
Introduction Human embryonic stem cells (hESCs) were isolated from the inner cell mass of blastocyst-stage embryos [1]. These cells can be propagated in culture for extended periods and can differentiate into all somatic cell types, underlying their great potential as a renewable source of functional cells for therapeutic applications. To realize this potential, however, it is necessary to devise robust and cost-effective systems for culturing undifferentiated hESCs at clinically relevant scales, and to develop strategies to direct the differentiation of hESCs to specific functional cell types. In this review we discuss issues specifically related to the development of robust systems for hESC propagation. We present advances in the development of culture systems for the maintenance of undifferentiated hESCs, discuss the current understanding of the molecular regulation of hESC selfrenewal, and identify key challenges that need to be addressed to facilitate the development of hESC-based therapeutic applications. Current Opinion in Biotechnology 2005, 16:568–576
Current hESC culture systems hESCs were originally derived in medium containing serum, with the support of feeder layers of mouse embryonic fibroblasts (mEFs) that secrete factors essential for maintaining hESCs in the undifferentiated state [1]. However, serum is a complex mixture of proteins of unknown composition, and different batches of serum exhibit variability. A defined culture system with feederfree conditions and medium of known protein composition is desirable to minimize the variability in culture conditions that affects the reproducibility and robustness of hESC culture. From the perspective of therapeutic applications, it might also be important for hESCs to be cultured (and derived) under xeno-free conditions (i.e. conditions free of components derived from animal sources). Indeed, hESCs cultured on feeders with medium containing serum components derived from animal sources express, at least transiently, an immunogenic nonhuman sialic acid and might not be immediately suitable for therapeutic applications [2]. The first feeder-layer-free cultures of hESCs used medium conditioned by the factors secreted by mEFs and MatrigelTM- or laminin-coated plastic plates [3]. MatrigelTM is a complex mixture of mouse sarcoma origin and contains mostly laminin, collagen IV, heparan sulfate proteoglycans (HSPGs) and entactin. Animal serum was replaced by a more defined serum replacement formulation called KnockOut Serum Replacement (SR) that contains components such as insulin, transferrin and lipid-rich bovine albumin [4]. Subsequently, the use of medium (containing SR) conditioned by hESC-derived fibroblasts (hEFs) immortalized by ectopic overexpression of telomerase to maintain hESCs in an undifferentiated state, was also reported [5]. This finding illustrates an important aspect of hESC culture that has to be considered during culture development — differentiated cells generated in non-supportive or poorly supportive hESC cultures might produce factors that promote or prolong hESC maintenance. Amit et al. [6] reported the maintenance of hESCs on a fibronectin matrix using unconditioned medium (i.e. medium containing SR but not conditioned by mEFs or hEFs) supplemented with basic fibroblast growth factor (bFGF), also known as fibroblast growth factor 2 (FGF2), and transforming growth factor-b1 (TGF-b1). However, hESCs cultured under these conditions have lower cloning efficiencies and growth rates and higher rates of differentiation than cells grown on mEF-conditioned media. HESCs have also been maintained in the undifferentiated state on MatrigelTM-coated plates in unconditioned medium www.sciencedirect.com
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Table 1 Feeder-free culture of human embryonic stem cells. Cell adhesion factor TM
Medium composition
Comments
Matrigel or laminin Human or bovine fibronectin
80% KO-DMEM; 20% KO-SR; mEF-CM + 4ng/mL bFGF 85% KO-DMEM; 15% KO-SR; 0.12 ng/mL TGF-b1; 4 ng/mL bFGF
MatrigelTM
80% KO-DMEM; 20% KO-SR; 24–36 ng/mL bFGF
MatrigelTM MatrigelTM
80% KO-DMEM; 20% KO-SR; 40 ng/mL bFGF 80% KO-DMEM; 20% KO-SR Medium conditioned by hES-derived fibroblasts, immortalized by ectopic overexpression of telomerase 80% DMEM/F12; 20% KO-SR; 500 ng/mL noggin; 40 ng/mL bFGF 80% KO-DMEM; 20% KO-SR; 50 ng/mL activin A; 50 ng/mL KGF; 10 mM nicotinamide
MatrigelTM Laminin
Lower cloning efficiency and growth rates and higher differentiation rates than cells grown on mEFs Different batches of bFGF required different working concentrations
Ref. [3] [6]
[8] [7] [5]
Higher concentrations of bFGF (100 ng/mL) can replace noggin
[9,10] [11]
In all cases, cell culture medium is supplemented with 0.1 mM b-mercaptoethanol, 1% non-essential amino acids stock and 1 mM (except in [6] where 2 mM L-glutamine was used). DMEM, Dulbecco’s modified Eagle medium; mEF-CM, SR-containing medium conditioned by mEFs.
L-glutamine
supplemented with high concentrations of bFGF [7,8], a combination of the bone morphogenic protein (BMP) antagonist noggin and bFGF [9,10] or a combination of keratinocyte growth factor (KGF, a member of the FGF family), nicotinamide and activin A [11]. A list of different culture systems reported for the maintenance of undifferentiated hESCs is presented in Table 1, highlighting the significant advances that have been made towards developing a defined culture system. However, components from animal sources have not been completely eliminated; SR and MatrigelTM both contain complex components of non-human origin. The use of human laminin to replace MatrigelTM could solve the problem in part, although significant variability exists in both human and mouse laminin obtained from different manufacturers and in different lots from the same manufacturer [9]. An interim solution to the problem of eliminating animal-derived components in hESC culture is the use of human feeders and human serum. Richards et al. [12] have demonstrated the derivation and propagation of a hESC line in completely xeno-free conditions. Table 2
summarizes the various sources of human feeders that have been validated for culture of undifferentiated hESCs [13–18]. Although a great deal of progress has been made in establishing better-defined conditions for the culture of hESCs, as is evident in Tables 1 and 2, the ultimate goal of a robust feeder-free system with a completely defined culture medium, where all components used are derived entirely from non-animal sources, remains elusive (see Update).
Extracellular factors regulating hESC self-renewal Work cited in the previous section indicates that the undifferentiated hESC state can be maintained by extracellular cues provided by the extracellular matrix (ECM) proteins, such as laminin, and exogenously added factors of the TGF-b and FGF families. The role of laminins in development has been discussed elsewhere [19]. Here we specifically examine the role of members of the TGF-b and FGF families in the regulation of hESC self-renewal.
Table 2 Culture of human embryonic stem cells on feeders from human sources. Feeder source
Comment
Ref.
Fetal muscle, fetal skin, adult fallopian tube Neonatal foreskin Adult skin, adult muscle Marrow stromal cells Adult uterine endometrial cells, adult breast parenchymal cells, embryonic fibroblasts Human embryonic stem cell line
Proof of derivation and propagation of a hESC line in completely xeno-free conditions
[12]
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A comparison of hES culture using human feeders from different sources hESCs grown on human uterine endometrial cells and embryonic fibroblasts have slightly different morphology (thinner and flatter) than cells grown on mEF An autogeneic feeder cell system is described. hESCs can also be cultured in feeder-free conditions using medium conditioned by autogeneic feeders.
[13,14] [15] [16] [17]
[18]
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The transforming growth factor-b superfamily
Signaling mediated by the ligands of the TGF-b superfamily has been previously reviewed by Shi and Massague [20]. Briefly, signaling is initiated by ligand binding to type I and type II receptors that have serine/threonine kinase activity. Ligand binding leads to phosphorylation of the type I receptor by the type II receptor and subsequent phosphorylation of Smad (mothers against decapentaplegic homolog [drosophila]) proteins by the type I receptor. The TGF-b superfamily ligands can be classified into two subfamilies: the TGF-b/activin/nodal subfamily that signals through the type I receptors activin receptor-like kinases (ALKs) ALK2, ALK4, ALK5, ALK7 and the receptor Smads (R-Smads) Smad2 and Smad3; and the BMP/growth and differentiation factor (GDF) subfamily that signals through the type I receptors ALK1, ALK3, ALK6 and the R-Smads Smad1, Smad5 and Smad8. Upon phosphorylation by the type I receptor tyrosine kinase, the R-Smads associate with a co-mediator Smad (C-Smad) Smad4 and translocate to the nucleus. The activated Smad complexes in the nucleus regulate the transcription of several target genes, in conjunction with other cofactors. Two inhibitory Smads (I-Smads), Smad6 and Smad7, negatively regulate signaling through the R-Smads. Smad7 inhibits all R-Smads, whereas Smad6 selectively inhibits Smad1. Gene expression analysis has shown that the genes associated with TGF-b and BMP signaling are expressed in hESCs [21,22]. The role of the BMP/GDF subfamily
Xu et al. [23] and Pera et al. [24] have reported two different effects of the BMP/GDF subfamily on hESCs. In the hESC line H1 cultured on MatrigelTM-coated plates with conditioned medium from mEFs, Xu and colleagues report differentiation of hESCs along the trophoblast lineage in response to BMP-4. A similar effect is observed in response to BMP-2, BMP-7 and GDF-5. In the case of hESC lines HES2 and HES3 cultured on feeders, the addition of BMP-2, BMP-4 or BMP-7 leads to differentiation to primitive endoderm [24]. Nevertheless, the undifferentiated hESC state is characterized by low levels of phosphorylated Smad1/5/8 [9,24,25], suggesting that suppression of BMP mediated signaling (i.e. inhibition of Smad1/5/8) is necessary for hESC maintenance. Indeed, medium conditioned by mEFs used in the maintenance of hESC cultures contains the BMP antagonists noggin and gremlin [9]. Also, hESCs can be maintained in the undifferentiated state in feeder-free conditions using unconditioned medium (medium containing SR but not conditioned by mEFs) supplemented with bFGF and noggin [9,10]. Some level of BMP signaling, however, might be important for maintenance of the undifferentiated hESC state. When noggin is added to hESC cultures on feeders, cells with neuroectodermal markers are obtained indicating that BMPmediated signaling might prevent differentiation to Current Opinion in Biotechnology 2005, 16:568–576
neuroectoderm [24]. This is consistent with data obtained using mouse embryonic stem cells where BMP-4 inhibits induction of neuroectodermal differentiation [26]. It is interesting to note the different effects of BMP on hESCs cultured on feeders (differentiation to primitive endoderm) from those cultured in feeder-free conditions with unconditioned medium (differentiation to trophoblast). One possible explanation for this difference, besides variability between cell lines used, is that the feeder layers provide other factors that inhibit BMP signaling. For instance, medium conditioned by mEFs contains activin A, which suppresses BMP signaling (elaborated further in the following section). Thus, the undifferentiated hESC state can be regulated in part through a balance between BMP signaling to inhibit neuroectodermal differentiation and the activity of BMP antagonists, such as noggin, involved in blocking BMP-induced differentiation to primitive endoderm and/ or trophoblast. The role of the TGF-b/activin/nodal subfamily
The undifferentiated hESC state is characterized by high levels of phosphorylated Smad2/3, indicating an important role for signaling mediated by the TGF-b/activin/ nodal subfamily in the regulation of hESC self-renewal [9,25,27]. SR-containing unconditioned medium exhibits BMP-like differentiation activity (either directly as a component of SR or indirectly through factors in SR that induce autocrine secretion of BMP) as seen by high levels of phosphorylated Smad1/5/8, detectable after 24 h culture with hESC, along with BMP-2 and BMP-4 proteins [9]. TGF-b1 [6] or activin A [11], together with bFGF, seem able to maintain undifferentiated hESCs under feeder-free conditions in SR-containing unconditioned medium. By themselves, in the same medium, TGF-b [6] or activin A [11] maintain the undifferentiated state, but cell proliferation is reduced. Also, medium conditioned by mEFs contains activin A [11]. Thus, the TGF-b/activin/nodal subfamily appears to suppress BMP signaling through Smad1/5/8. As discussed earlier, because BMP signaling drives differentiation to trophoblast and/or primitive endoderm, the TGF-b/activin/ nodal subfamily presumably suppresses differentiation to these lineages. Signaling through Smad2/3 leads to increased expression of nodal and the nodal antagonists lefty-A and lefty-B [27]. Nodal is known to prevent differentiation of hESCs along the neuroectodermal pathway [28]. Downregulation of nodal, lefty-A and lefty-B gene expression is one of the earliest events upon differentiation induced by withdrawal of conditioned medium [27]. Nodal can bind BMP in the extracellular milieu, leading to inhibition of the effects mediated by both nodal and BMP [29]. Thus, Smad2/3 signaling negatively regulates signaling through Smad1/5/8 and the effect of nodal (and hence Smad2/3) through expression of the lefty proteins. www.sciencedirect.com
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The fibroblast growth factor family
The other major player in regulating hESC self-renewal is the fibroblast growth factor (FGF) family, extensively reviewed in [30–32]. The FGF family consists of 22 ligands signaling through four cell-surface FGF receptors (FGFR1–4) with intrinsic tyrosine kinase activity. Alternative splicing leads to numerous isoforms of FGFR. Each FGFR can bind to multiple FGF ligands. In addition, HSPGs act as low-affinity receptors for the FGF ligands and stabilize the formation of the FGF–FGFR complex. FGF binding to FGFR induces receptor dimerization and initiates tyrosine kinase activity and subsequent activation of the Ras-mitogen associated protein kinase (MAPK), phosphatidylinositol-3 (PI3) kinase and phospholipase C-g (PLC-g) pathways. Gene expression analysis indicates that FGF signaling is active in undifferentiated hESCs [21,22] (see Update). Indeed, bFGF is an important exogenously added factor in the medium used to culture undifferentiated hESCs. In the absence of bFGF, the undifferentiated hESC state can be maintained in unconditioned medium by TGF-b1 or activin but the cells proliferate poorly [6], suggesting that bFGF plays a role in hESC mitogenesis [33]. Also, signaling through the PI3 kinase/Akt/protein kinase B (PKB) pathway initiated by bFGF has been implicated in the expression of ECM molecules [34]. High concentrations of bFGF can replace noggin in maintaining the undifferentiated hESC state in unconditioned medium, thus suppressing the BMP-like differentiation activity induced by unconditioned medium [9]. However, under these conditions, phosphorylation of Smad1 is still observed. A likely explanation for this phenomenon is that MAPK activated by bFGF signaling prevents nuclear localization of phosphorylated Smad1 (explained in the following section) [35].
Interactions regulating the undifferentiated hESC state Self-renewal of hESCs is regulated by exogenously added and autocrine factors the interact with each other and the cells in the extracellular milieu. Interactions between these factors in an extracellular context and the crosstalk between the intracellular signaling pathways activated by them contribute to maintenance of the undifferentiated hESC state. Figure 1a illustrates the balance between different known factors in the extracellular milieu that maintain the hESC in the undifferentiated state; Table 3 is a concise compilation of the information used to construct Figure 1a. Important aspects of the crosstalk between the signaling pathways activated by these extracellular factors is shown in Figure 1b. Outside the cell, binding interactions between BMP and noggin [9,10] and BMP and nodal [29] negatively regulate BMP signaling. Binding between BMP and nodal also inhibits nodal signaling [29]. The nodal antagonists lefty-A and lefty-B inhibit nodal signaling by www.sciencedirect.com
directly interacting with nodal, thereby preventing binding to its receptor complex or interacting with the EGF-CFC proteins (accessory receptors for nodal) and preventing formation of the nodal receptor complex [36]. HSPGs present on the cells as well as in the ECM bind to FGF and can regulate FGF signaling. Intracellularly, significant cross-talk exists between the FGF and TGF-b/BMP signaling pathways. The R-Smads contain an N-terminal MH1 domain and a C-terminal MH2 domain. Phosphorylation of the C-terminal MH2 domain by the type I receptor kinase is necessary for activation of the R-Smads [20]. The linker region between the MH1 and MH2 domains also contain phosphorylation sites that mediate crosstalk between the Smad and MAPK signaling pathways. Phosphorylation of the linker region of R-Smads by MAPK alters the nuclear localization and hence activity of the R-Smads. Linker phosphorylation of Smad1 and Smad2 prevents nuclear localization, whereas linker-phosphorylated Smad3 is localized in the nucleus [35,37]. Thus, the biological activity of the R-Smads is regulated by MAPK signaling. Members of the TGF-b superfamily, bFGF and the lefty proteins can all activate MAPK signaling [20,30,32,38]. The phosphorylation of Smad3 at the C-terminal MH2 domain is inhibited by direct interaction with Akt/PKB [39,40]. As discussed earlier, FGF mediates signaling through the PI3 kinase/Akt/PKB pathway. Gene expression analysis suggests that the Wnt signaling pathway might be active in undifferentiated hESCs [22], although the role of Wnt in hESC maintenance is unclear. The hESC undifferentiated state and corresponding high levels of phosphorylated Smad2/3 can be maintained in unconditioned medium, at least over short periods of time, in the presence of a glycogen synthase kinase 3-b (GSK3-b) inhibitor [25,41]. The level of phosphorylated Smad2/3 decreases in the presence of an inhibitor that prevents phosphorylation of Smad2/3 by the type I TGF-b receptors, suggesting that the inhibition of GSK3b is associated with expression of factors that act in the extracellular milieu and are involved in Smad2/3 signaling [25]. Cross-talk also exists between the FGF and Wnt signaling pathways [30]. Akt/PKB phosphorylates GSK3b leading to inhibition of GSK3-b activity. It is interesting to note that the Wnt, FGF and TGF-b signaling pathways converge at GSK3-b, thereby implicating it as a convergence node in these interacting signaling networks. Together, these signaling results support an emerging perspective that hESCs, like mESCs [26] (and perhaps stem cells in general), maintain their undifferentiated state by inhibiting signaling (and gene expression) programs that lead to the development of specific cell types. At the signaling level, interactions between extracellular factors and crosstalk between signaling pathways may be an important part of this negative regulation. It is Current Opinion in Biotechnology 2005, 16:568–576
572 Biochemical engineering
Figure 1
Molecular interactions regulating hESC self-renewal. (a) Extracellular factors regulating hESC self-renewal and their interactions. Red lines indicate negative regulation; green arrows show positive regulation. Solid lines denote extracellular interactions; dashed lines indicate intracellular interactions or indirect effects. (b) Cross-talk between intracellular signaling pathways regulating hESC self-renewal. Red lines indicate negative regulation; green arrows show positive regulation. Solid lines denote direct interactions; dashed lines indicate indirect effects.
particularly appealing, therefore, to consider the ESC as an unstable steady state [42] (as shown in Figure 2) balanced in a high energy local minimum in the energy landscape, with a tendency upon perturbation to differentiate (to a lower energy state) along different lineages. Understanding this balance, as well as how to systematically perturb it, will be an important aspect of hESC research in the next few years.
Near-term challenges in hESC culture development Recent advances in stem cell biology have led to a clearer definition of the players involved in hESC self-renewal and the concomitant development of better-defined conditions for undifferentiated hESC culture. However, current understanding is far from perfect and several key Current Opinion in Biotechnology 2005, 16:568–576
challenges need to be overcome for the culture of undifferentiated hESCs in a therapeutically relevant manner. A major problem in the design of bioprocesses for hESC propagation is the need for the development of rapid, robust and predictive assays for screening the impact of culture manipulations on hESC maintenance. The classic definition of the ESC state is largely based on the mESC paradigm. Simplistically, an ESC (as defined in the context of the mESC) can be propagated extensively in culture, has a normal diploid karyotype, and can give rise to cells of all embryo-derived lineages in vivo and in vitro. In hESCs, the ability of the cells to recapitulate all aspects of in vivo embryogenesis cannot be validated. Currently a hESC culture system is considered supportive if the cells retain cell surface and intracellular markers associated www.sciencedirect.com
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Table 3 Effect of different factors on the regulation of human embryonic stem cell self-renewal. Factor
Effects
Ref.
BMP
BMP promotes differentiation to trophoblast BMP promotes differentiation to primitive endoderm Noggin promotes self-renewal by antagonizing BMP Presence of noggin leads to the upregulation of neuronal markers Therefore, BMP prevents differentiation to neuroectoderm
[23] [24] [7,10] [24] (See update)
Nodal
Nodal prevents differentiation to neuroectoderm Activation of Smad2/3 by nodal leads to expression of nodal and the nodal antagonists lefty-A and lefty-B Nodal binds to and inhibits BMP
[28] [27]
Activin A
Activin A can maintain hESC undifferentiated state in the presence of FGF Inhibition of BMP signaling is necessary to maintain hESC undifferentiated state Therefore, activin A inhibits the effect of BMP signaling
[11] [9,10]
TGF-b
TGF-b can maintain undifferentiated hESC state in the presence of FGF Inhibition of BMP signaling is necessary to maintain hESC cell undifferentiated state Therefore, TGF-b inhibits the effect of BMP signalling
[6,11] [9,10]
bFGF
High concentrations of bFGF can replace noggin to maintain the undifferentiated hESC state Therefore, BMP signaling is antagonized by bFGF
[9]
[29]
Statements in italics are inferences deduced from the references cited.
with (and previously defined to be correlated with) hESC pluripotency, are karyotypically normal, and are able to differentiate into (functional) cells of ectoderm, mesoderm and endoderm lineages — a property typically detected in vitro using embryoid body formation assays and/or in vivo using teratoma formation assays in mice (reviewed in [43]). Thus, the currently accepted paradigm for assaying the pluripotency of hESCs involves tedious and lengthy experimental procedures and does not typically allow for the interrogation of hESC properties at the clonal level (a criterion necessary to rigorously establish quantitative relationships between the microenvironment and stem cell maintenance). A rapid assay that uses quantifiable parameters such as concentration or the behavior of particular molecular species and reliably predicts individual hESC developmental potential is highly desirable. Several studies report the use of gene
expression analysis to identify a set of genes that are differentially expressed in pluripotent hESCs, and hence assign a unique molecular signature to undifferentiated hESCs [44,45] (see Update). In the mESC system, Palmqvist et al. [46] reported the correlation of gene expression profiles using functional assays that assess developmental potential to identify genes that are downregulated during early differentiation. However, in the case of hESCs, similar studies that compare gene expression analysis to functional measures of pluripotency (hence validating a putatively unique signature) have not been reported. An alternate approach might be to use hESC signaling responsiveness to define a unique molecular signature for undifferentiated hESCs. Understanding unique aspects of static or dynamic hESC signaling networks will be important if this approach is to prove fruitful.
Figure 2
The hESC modeled as an unstable steady state. Different factors maintain the steady state by preventing differentiation along different paths (e.g. noggin and bFGF prevent differentiation to the trophoblast by blocking BMP signaling). www.sciencedirect.com
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574 Biochemical engineering
As discussed earlier, several media formulations have been used for culturing undifferentiated hESCs; however, hESC culture systems predominantly use MatrigelTM as the ECM substrate for cell attachment. The development of engineered attachment substrates for the culture of hESCs remains an important challenge. Understanding the interactions between signaling pathways initiated by the cytokines in the medium and signaling associated with cell adhesion and spreading on the ECM will facilitate the design of improved attachment substrates [47]. Anderson et al. [48] have developed a highthroughput system for synthesizing and evaluating novel substrates for surface attachment of hESCs. The use of such high-throughput platforms (and those described elsewhere [49]) in conjunction with predictive screening-based assays for hESC developmental potential could be an effective approach for hESC culture development. Finally, defined and robust culture systems will have to be integrated with cost-effective strategies for the generation of significant numbers of validated hESCs under good manufacturing practice (GMP) conditions. Microenvironmental parameters such as dissolved oxygen, pH and media supplementation play an important role in ESC culture (see Update). For instance, dissolved oxygen affects both ESC self-renewal and differentiation; low oxygen tension has been shown to prevent the differentiation of hESCs [50]. In the mESC system, hypoxic conditions improve the yield of ESC-derived cardiomyoctes [51]. Low levels of oxygen are likely to affect ESC signaling pathways through the hypoxia-response element inducible proteins. In mESCs, survival under prolonged hypoxic conditions (48 h) is mediated by autocrine vascular endothelial growth factor, which is upregulated by hypoxia-inducible transcription factors [52]. A systematic analysis of the impact of the dynamic interactions between culture variables such as oxygen tension and intracellular signaling that controls ES cell fate will be important for the development of robust stem-cell technologies. The development of bioreactors based on an in-depth understanding of controllable, seemingly pedestrian but nonetheless critical, microenvironmental parameters will be required for the generation of therapeutically useful hESCs.
specific differentiated cell types from hESCs, a multistep process that might involve tipping the balance between contrasting cell fates at multiple steps along the way.
Update A demonstration of a defined and xeno-free culture system for undifferentiated hESCs has recently been reported by Li et al. [53]. hESCs were cultured on surfaces coated with laminin of human placental origin, using a commercially available defined serum-free medium that does not contain animal-sourced products, supplemented with high concentrations of human bFGF. The role of bFGF in hESC culture has been further elaborated in an analysis that suggests a direct influence of signaling mediated by endogenous bFGF on hESC self-renewal [54]. In another recent study, Yang et al. [55] have reported the development of a focused microarray that will assist in evaluating the effect of culture microenvironment on hESC self-renewal and differentiation. Gerrard et al. [56] report hESC differentiation to neural lineages, in attached cultures, upon blocking of BMP signaling by noggin. The importance of cellular microenvironment in hESC culture is further demonstrated in a recent study by Saha et al. [57], which shows that the mechanical agitation of the cell substratum results in the inhibition of hESC differentiation relative to unmanipulated controls.
Acknowledgements We apologize to our colleagues whose work was not cited in this review. Thanks to Ryan Davey and Jonathan Draper for helpful comments. BMR is supported by the Canadian Stem Cell Network and the Stem Cell Biology and Regenerative Medicine Center at the Robarts Research Institute (London, Ontario). PWZ is the Canada Research Chair in Stem Cell Bioengineering.
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