Ava i l ab l e on l i ne a t www.sc i enced i r ec t . com

ScienceDirect

www.e l sev i e r . com / l oca te / s c r

Stem Cell Research (2014) 12, 610–621

Developmental insights from earlymammalian embryos and core signalingpathways that influence human pluripotentcell growth and differentiation

Kevin G. Chena,⁎, Barbara S. Mallona, Kory R. Johnsonb,Rebecca S. Hamiltona, Ronald D.G. McKayc, Pamela G. Robeyd

a NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda,MD 20892, USAb Information Technology and Bioinformatics Program, National Institute of Neurological Disorders and Stroke,National Institutes of Health, Bethesda, MD 20892, USAc The Lieber Institute for Brain Development, 855 North Wolfe Street, Baltimore, MD 21205, USAd Craniofacial and Skeletal Diseases Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health,Bethesda, MD 20892, USA

Received 26 September 2013; received in revised form 10 January 2014; accepted 4 February 2014Available online 19 February 2014

Abstract Human pluripotent stem cells (hPSCs) have two potentially attractive applications: cell replacement-basedtherapies and drug discovery. Both require the efficient generation of large quantities of clinical-grade stem cells that are freefrom harmful genomic alterations. The currently employed colony-type culture methods often result in low cell yields,unavoidably heterogeneous cell populations, and substantial chromosomal abnormalities. Here, we shed light on the structuralrelationship between hPSC colonies/embryoid bodies and early-stage embryos in order to optimize current culture methodsbased on the insights from developmental biology. We further highlight core signaling pathways that underlie multipleepithelial-to-mesenchymal transitions (EMTs), cellular heterogeneity, and chromosomal instability in hPSCs. We also analyzeemerging methods such as non-colony type monolayer (NCM) and suspension culture, which provide alternative growth modelsfor hPSC expansion and differentiation. Furthermore, based on the influence of cell–cell interactions and signaling pathways,we propose concepts, strategies, and solutions for production of clinical-grade hPSCs, stem cell precursors, and miniorganoids,which are pivotal steps needed for future clinical applications.

Published by Elsevier B.V. Open access under CC BY-NC-ND license.

Abbreviations: FGF, fibroblast growth factor; hPSCs, human pluripotent stem cells; hESCs, human embryonic stem cells; hiPSCs, humaninduced pluripotent stem cells; NCM, non-colony type monolayer; ROCK, Rho-associated kinase; TGF-β, transforming growth factor β⁎ Corresponding author at: NIH Stem Cell Unit, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 37

Convent Drive, Room 1000, Bethesda, MD 20892, USA.E-mail address: cheng@mail.nih.gov (K.G. Chen).

http://dx.doi.org/10.1016/j.scr.2014.02.0021873-5061/Published by Elsevier B.V. Open access under CC BY-NC-ND license.

611Pluripotent stem cell growth models

Introduction

Human pluripotent stem cells (hPSCs), including humanembryonic stem cells (hESCs) and induced pluripotent stemcells (iPSCs), are potentially important resources for regener-ative medicine-based cell replacement. However, this ap-proach has not been efficiently implemented, due in part tocurrently used inefficient cell culture and differentiationstrategies, which are heavily based on cell culture as coloniesand aggregated embryoid bodies (EBs) (Chen et al., 2014;Mallon et al., 2006; Thomson et al., 1998; Xu et al., 2001).These methods have led to time-consuming production ofrelatively low numbers of cells (Hartung et al., 2010; Kehoeet al., 2010; Serra et al., 2012), heterogeneous cellular andmolecular states (Bendall et al., 2007; K.G. Chen et al., 2012;Moogk et al., 2010; Stewart et al., 2006), and frequent reportsof chromosomal abnormalities (reviewed in Baker et al., 2007;Lee et al., 2013). Certain chromosomal alterations such astrisomies 12, 17, and 20 have the capacity to confer growthadvantages as indicated by comparative genetic analyses(Amps et al., 2011; Catalina et al., 2008; Draper et al., 2004;Lefort et al., 2008).

The relationship between heterogeneous cellular statesand the incidence of chromosomal abnormalities remainsunclear. Thus far, there is no direct proof that the colony-type culture method is responsible for the reported chromo-somal abnormalities. However, it is conceivable that theheterogeneity of hPSC colonies (induced by cellular stress)might result in the selection of cells with growth advantages,which are usually bestowed by mutations or chromosomalalterations (Baker et al., 2007; Lee et al., 2013). This cellularstress may be derived from unwanted growth factors orconditions, which could be categorized as abnormal metabolicpathways, deficiency in nutrient or growth factors, andexcessive apoptotic or differentiation signals. These subop-timal conditions may directly or indirectly contribute toinsufficient cell production, cellular heterogeneity, andchromosomal instability of hPSCs.

It is imperative that we develop efficient culture methodsthat can overcome the obstacles found in the current culturesystems. Moreover, new culture methods should be optimizedfor production of clinically relevant cell numbers, tissuemorphogenesis, high-throughput drug discovery, and effi-cient assays of cellular stress. A comprehensive analysis ofdifferent cell culture protocols for hPSC is available in arecent review (Chen et al., 2014). Here, we will focus on thefundamental principles of developmental biology and coresignaling pathways that underlie different growth methods.We will further discuss the relationship between the develop-ment of early stage embryos and the mechanisms underlyinghESC heterogeneity and chromosomal instability. Finally, wewill provide new insights into the development of optimalgrowth models for hPSCs.

Human pluripotent stem cell colonies andaggregates recapitulate some properties ofearly-stage embryo development

Unlike mouse embryonic stem cells (mESCs), which possesshigh single-cell plating efficiency under dissociated conditions,

both hESCs and iPSCs cannot grow as single cells undercommonly used culture conditions. Human pluripotent stemcells are conventionally cultured as condensed colonies andmust be passaged as cell clumps to avoid cumulative apoptoticand differentiation stress. These cells are routinely differ-entiated toward derivatives of the three germ layers throughthree-dimensional (3D) EBs. In general, colony-type andaggregated cultures often produce heterogeneous coloniesand EBs. Hence, we frequently observe more apoptoticactivity in the center of a colony (with more spontaneouslydifferentiated cells at the periphery) (Figs. 1 and 2) andcomplicated cystic structures after EB agglomeration (Fig. 3).The unique structures of hESC colonies and EBs are reminis-cent of the inner cell mass (ICM) and post-implantationblastocysts observed during early embryo development. Inthe following sections, we will discuss the links between thestructures of hPSC colonies and EBs and their developmentalcounterparts.

Human ES colony structure inherits epithelial featuresof the ICM

The ICM is comprised of a group of epithelial-like cells, theepithelial features of which are reflected at the intercellularjunctions and in their basement membranes (Fig. 1A). Typicalepithelial cells are tightly polarizedmonolayers with a distinctapical surface, basal membrane, and lateral intercellular con-junctions (Figs. 2A, B). At day 4 of compacting humanembryos, E-cadherin (E-cad), an important component ofepithelial cells, is located on the membranes where cell–cell contacts occur. However, in the ICM, E-cad is diffuselyexpressed in the cytoplasm, but not on the plasmamembrane (Alikani, 2005). Thus, the distribution of E-cadin human preimplantation embryos appears to be an embry-onic stage-dependent process. Electron microscopic study hasalso revealed similar epithelial features in hESCs (Ullmannet al., 2007). Intercellular interactions in both trophectodermand ICM cells are implemented through gap junctions, tightjunctions, and desmosome-like structures (Dale et al., 1991;Gualtieri et al., 1992).

Laminin is another epithelial component of the basementmembrane that is expressed in the different stages of earlyembryos. Matrigel, a basement membrane preparation ex-tracted from a murine Englebreth–Holm–Swarm sarcoma(Kleinman et al., 1982), containing laminins, as well ascollagen IV, fibronectin, and vitronectin, was found to supportrobust hESC growth in defined medium (Ludwig et al., 2006)and is used routinely for the feeder-free culture of hPSCs.Laminin-111, the most abundant form in the Reichert'smembrane, was found under the mouse trophoblasts, whichwas proposed to interact with ICM cells and induce differen-tiation (Domogatskaya et al., 2012; Miner et al., 2004). Atleast two laminin isoforms (i.e., laminin-511 and -521) havebeen detected in the basement membrane of the ICM ofmammalian pre-implantation embryos (Domogatskaya et al.,2012; Miner et al., 2004). It has been shown that laminin-511,but not -111, supports mESC self-renewal in vitro withoutleukemia inhibitory factor (LIF) or mouse embryonic fibro-blasts (MEFs) (Domogatskaya et al., 2008). Recently, the useof human recombinant laminin-511 has enabled long-termself-renewal of hPSCs (Rodin et al., 2010). Thus, both E-cad

Figure 1 Early human embryo development, hESC culture, and acquisition of cellular heterogeneity. (A) Anatomical structure ofthe inner cell mass (ICM) at day 8 with major cell types indicated. (B) Derivation and growth of hESCs from the ICM: A typical hESCcolony (e.g., hESCs grown on feeders) has three distinct zones in terms of its growth patterns, cell morphology, densities, differentialmarker expression, and differences in response to small molecules and signaling factors. The center of a colony represents highlycompacted epiblast-like cells (EpiBL), which frequently experience either apoptotic or differential stress. The periphery is the regionthat undergoes epithelial-to-mesenchymal transitions (EMTs), whereas some differentiated fibroblast-like cells (FBL) expressdifferential markers SSEA-1 and ABCG2 and are sensitive to BMP-4 signaling (K.G. Chen et al., 2012; Padmanabhan et al., 2012). Theregion between the center and periphery of the colony represents an intermediate state of cells that undergo reversible EMTs.Undifferentiated hESCs could be selected or adapted after serial passaging through growth-differentiation selection cycles in in vitrogrowth conditions. (C) Lineage commitment from blastocyst (BC) to gastrulation, trophectoderm (TE), and extraembryonic endoderm(EEE). The flow chart depicts the relationship among the cell types presented in panel A. EMT occurs at the primitive streak.(D) Anatomical structure and various cell types in hESC colonies under colony-type culture conditions: hESCs undertake eitherreversible EMT (that maintain the undifferentiated states) or irreversible EMT (that induce terminal differentiation). Abbreviations:BC, blastocyst; BM, basement membrane; CTB, cytotrophoblast; EEE, extraembryonic endoderm; EEM, extraembryonic mesoderm;EM, endometrium; EpiB, epiblast; EpiBL, epiblast-like cells; FBL, fibroblast-like cells; HB, hypoblast; HBL, hypoblast-like cells; ICM, innercell mass; TE, trophectoderm; STB, syncytiotrophoblast; TB, trophoblast; and TBL, trophoblast-like cells.

612 K.G. Chen et al.

and some specific laminins are indispensable inmaintaining theepithelial features of undifferentiated hESCs. Alterations ofthese epithelial components in vitro during embryonic devel-opmentmay induce differentiation and cellular heterogeneity.

Figure 2 Growth models of hPSCs under various growth conditiocontains growth factors FGF2 and TGFβ on Matrigel-based extracellucomplexes: Laminin (LM), fibronectin (FN), and vitronectin (VNT)respectively (Braam et al., 2008; Brafman et al., 2012). Diffeepithelial-to-mesenchymal transition (EMT) states, in which thecharacteristics and cells at the periphery develop mesenchymal-likethe colonies define an intermediate state that undergoes transient(e.g., E-cad) with degradation of ECM confers cells with an irreversibGrowth models of hPSCs grown as non-colony type monolayer (NCM)conditions as indicated in panel A. (C) Core pathways that support hPamong epithelial hPSCs, cells with an intermediate ES state, and diEMT2) or METs (MET1 and MET2) are likely to mediate these converEMTs and METs are proposed and indicated with an emphasis on the dv-akt murine thymoma viral oncogene homolog; ALK5, transformingkinase 5; BM, basement membrane; CDH1, the gene encoding E-cadextracellular signal-regulated kinase; FGF2, fibroblast growth factoINT, intergrins; LM, laminin; MAPK, mitogen-activated protein kinmetallopeptidases; Nu, nucleus of the cell; p53, tumor protein p53;culture dishes for hPSCs; RTK, receptor tyrosine kinases; Smad, SMAhomolog 2 (Drosophila); TGFβ, transforming growth factor beta; VN

Within the ICM, the embryoblast has the ability to form theepiblast, the outer layer of a blastula that gives rise to theectoderm, and the hypoblast, which in turn generates extraem-bryonic endoderm (Fig. 1C) (Carlson, 2009). Developmentally,

ns. (A) Growth models of hPSC colonies in hESC medium thatlar matrices with deduced anatomical structures and molecularmediate their effect via the α6β1, α5β1, and αVβ5 integrinsrent zones of out-growing hPSC colonies have differentialcells in the center of the colonies possess more epithelialphenotypes. The cells between the center and the periphery ofEMT and sustains pluripotency. Loss of the epithelial markersle EMT state with fibroblast- or trophoblast-like phenotypes. (B)have uniform intercellular interactions under the same growthSC growth: the model illustrates the dynamics of cellular statesfferentiated cells, in which two phases of EMTs (i.e., EMT1 andsions. The potential regulators and pathways that control bothifferential roles of TGFβ at different stages. Abbreviations: AKT,growth factor, beta receptor 1, known as activin receptor-likeherin (epithelial); ColV, collagenase IV; E-cad, E-cadherin; ERK,r 2 (basic); FN, fibronectin; GSK3, glycogen synthase kinase 3;ase; MET, mesenchymal-to-epithelial transition; MMPs, matrixPPJ, plastic-protein junction; PS, polystyrene surface of the cellD family member; Snail, snail homolog 1 (Drosophila); Slug, snailT, vitronectin; β-cat, β-catenin.

613Pluripotent stem cell growth models

embryonic epithelial cells can change their morphology andmotility to resemble fibroblast-like mesenchymal cells, a processknown as epithelial-to-mesenchymal transition (EMT) (Carlson,2009). The peripheral cells of the embryoblast differentiate into

the cytotrophoblast and syncytiotrophoblast, resembling theEMTs required for implantation (Fig. 2A). The reverse process ofEMT, termedmesenchymal-to-epithelial transition (MET), can berepeatedly observed during normal embryonic development

Figure 3 Early mammalian embryo development and embryoid body (EB) differentiation. (A) Structural presentation of mammalianembryonic differentiation after the implantation of a blastocyte. Syncytiotrophoblasts are not shown in this figure. The figure isadapted from the book Human Embryology and Developmental Biology (Carlson, 2009). (B) Delineation of early- and late-stage EBformation with differentiation medium in a non-adherent culture dish. Abbreviations: CTB, cytotrophoblast; EpiB, epiblast; EpiBL,epiblast-like cells; HB, hypoblast; HBL, hypoblast-like cells; ICM, inner cell mass; and TB, trophoblast.

614 K.G. Chen et al.

(Carlson, 2009). Thus, hESCs derived from the ICM may beconsidered to mimic epiblast-stage embryos before the occur-rence of the primitive streak, where the first EMT event takesplace (Figs. 1C, 2C). It is clear now that multiple phases of EMTand MET play pivotal roles in early embryo development and inthe generation of various types of tissues (Carlson, 2009; Thieryet al., 2009).

It is feasible that hESC lines, derived from the ICM, havepredominantly epithelial characteristics and, to a lesser extent,resemble cell types with mesenchyme-like propensity. Consis-tently, expression of the epithelial marker genes CDH1, DSP andKRT19 is significantly increased in hESCs, as opposed to thatobserved in fibroblasts and partially reprogrammed iPSCs(Ullmann et al., 2007). Moreover, EMT has also been found incolonies of monkey ESCs and in feeder-free cultures thatfrequently induce spontaneous differentiation (Behr et al.,2005; Pratama et al., 2011). Thus, the heterogeneity of a hESCcolony might be an inherent property of the ICM, which at leastis partially attributed to irreversible EMTs (Figs. 1 and 2). It isconceivable that the heterogeneity of the ICM is required forconsecutive three-germ layer differentiation after embryoimplantation. In vitro, the counterpart of the three germ-layerlineage commitment is EB-mediated differentiation.

EB differentiation shares some fates of implantedblastocysts

Without growth factors that support the pluripotent state,hESC clumps form 3D multilayer aggregates under suspensionculture conditions, usually in a non-adherent culture dish.EB differentiation has been widely used as an intermediatestep in multilineage differentiation. As the name suggests,these aggregates mimic many aspects of cell differentiationduring the development of early embryos (Fig. 3). Based on

the stage and structural properties, EBs can be classified intosimple and cystic EBs (Fig. 3B) (Magyar et al., 2001; Ungrinet al., 2008). Simple EBs are 2- to 4-day spherical aggregates(usually 100–400 μm), resembling morula-like and, to someextent, the ICM-stage embryos. With respect to cystic EBs,there are one ormore central cavities after day 4 in suspensionculture, which are similar to an implantedmammalian embryofrom day 7 to 12. At this stage, the embryos have establishedan inner parietal endoderm enclosed cavity (i.e., primary yolksac) and an outer layer termed extraembryonic mesoderm(Fig. 3A) (Carlson, 2009).

EBs are developed through intercellular adhesion, whichmay involve the cadherin–β-catenin–Wnt pathway, a potentregulatory mechanism for tissue morphogenesis (Gumbiner,1996). Indeed, E-cad, expressed in undifferentiated hESCs, isthe initiator of early-stage EB formation and also plays a role inlater-stage EB differentiation (Dang et al., 2004). Modulationof a specific cadherin isoform is associated with a definite celllineage commitment, in which expression of E-cad leads toepithelial differentiation whereas up-regulation of N-cadprefers a neuroepithelial fate (Twiss and de Rooij, 2013).Moreover, spontaneous formation of primitive endoderm wasobserved at the exterior surface of EBs after cellaggregation (Maurer et al., 2008). The primitive endodermcells were subsequently differentiated into visceral and parietalendoderm, and also generated a laminin–collagen-IV-enrichedbasement membrane (Fig. 3B) (Li et al., 2001). Similarly, theexterior domain of hESC EBs was stained positively for theendoderm marker FOXA2 (Ungrin et al., 2008). Unlike theirembryonic counterpart, regular EBs do not have a thin layer oftrophoblasts (Fig. 3). Thus, the exterior surface is directlyexposed to soluble factors and nutrients in culture medium.

The permissive diffusion of soluble factors (b1000 Da)in culture medium from the exterior to the interior ofEBs enables the formation of concentration gradients that

615Pluripotent stem cell growth models

influence the differentiation outcomes (Bratt-Leal et al.,2009). As EB development proceeds, asynchronous differ-entiation of EBs toward the three germ-layer lineagesmay be achieved. However, this heterogeneous differentiationpresents a big challenge for large-scale production of homoge-neously differentiated cells. Numerous efforts were made tocontrol the size of both hESC colonies and aggregates in orderto direct differentiation trajectories (Bauwens et al., 2008). Toensure the homogeneity of EBs, Zandstra and colleagues alsoutilized a new technique to generate uniform EBs fromdissociated single-cells. The size-controlled EB formation withthe ROCK inhibitor Y-27632 ensured its homogeneity and highlyordered structures (Ungrin et al., 2008). In addition, strategiesthat influence differentiation from both the exterior andinterior of EBs can be used to efficiently direct differentiationby controlling the extracellular microenvironment andchemical signals (Bratt-Leal et al., 2009).

Collectively, EB-based methods may offer several benefitsfor stem cell research, which include (i) use of EBs as a routinedifferentiation protocol, (ii) application of EBs to study earlydifferentiation events of human embryos, (iii) use of geneti-cally engineered EBs to determine the functionality of genemutations that are lethal to early embryos, (iv) coupling ofEB methods with new growth modules such as bioreactors toproduce large amounts of differentiated cells for therapeu-tic use, and (v) combination of EB methods with microfluidicbioreactors to study morphogenesis in 4D culture systems(Chen et al., 2014). Nevertheless, we also need to considerthe disadvantages of EB methods (i.e., time-consumingnature, cell loss, lower differentiation efficiency, and hetero-geneous differentiation) in future pharmaceutical and clinicalapplications.

Thus, both colony-type hPSC growth and EB aggregateddifferentiation share many structural features of early embry-onic development, including the heterogeneous nature ofcell growth and differentiation. It has been appreciatedthat generation of heterogeneity is actually required forlineage commitment and tissue maturation (Carlson, 2009).However, such a heterogeneous cellular state is an unwantedoutcome for hPSC-based therapies, which usually need trans-plantation of a pure cell population. Understanding the precisesignaling pathways that drive early embryonic developmentand hESC growth would provide useful tools to control theseheterogeneous states during cell expansion and differentiationin vitro.

Core signaling pathways depict multiple EMTphases and cellular heterogeneity in humanpluripotent stem cells

The molecular mechanisms that maintain the epithelialidentity of hPSCs depend on apical ligand-receptor signaltransductions, intercellular adhesion (mediated by moleculessuch as E-cad-conjugated β-catenin), and basement mem-brane and ECM interactions (Figs. 2A, B). With regardto intercellular connections, glycogen synthase kinase-3(GSK-3) is a potent regulator of E-cad and plays a pivotalrole in EMT and in cell fate commitment. In general, GSK-3phosphorylates Snail and initiates its proteasomal degrada-tion, hence upregulating E-cad and concurrently inhibitingEMT (Doble and Woodgett, 2007). However, in the case of

hESCs, when GSK-3 inhibitor (6-bromoindirubin-3′-oxime) isadded as a supplement to hESC medium to support hESCself-renewal, EMT is inhibited, likely through activation ofthe Wnt pathway (Sato et al., 2004; Ullmann et al., 2008).Nevertheless, the role of Wnt/β-catenin in the main-tenance of undifferentiated hESCs has been questionedby a recent study in which the authors revealed thatWnt/β-catenin signaling actually promotes hESC differentia-tion, not self-renewal (Davidson et al., 2012). In general,concurrent upregulation of E-cad and down-regulation ofmetallopeptidases (such as MMP2 and MMP9), N-cadherin,vimentin, Snail, and Slug may be essential to inhibit EMT anddifferentiation (Fig. 2C) (Ullmann et al., 2008).

Interestingly, E-cad may coordinate with many molecularevents to reduce the motility and the occurrence of thefibroblast-like cells at the periphery of hESC colonies invarious growth conditions (Ullmann et al., 2008; Ullmannet al., 2007). E-cad may be also associated with thearrangements of the core actin cytoskeleton and the EMTphenotype. This E-cad-EMT reverse correlation may beassayed by the presence of the membrane oncofetal protein5T4, which is triggered by the addition of anti-adhesiveantibody SHE78.7 that disrupts E-cad (Eastham et al., 2007).These data suggest that loss of E-cad and its regulators is acritical step that initiates EMT in hESCs.

Several other signaling pathways that include the TGFβand phosphoinositide 3-kinase (PI3K) pathways have alsobeen implicated in the regulation of EMT. However, the roleof TGFβ in EMT (the differentiation vector) and maintenanceof undifferentiated hESCs is somewhat complicated. It isbelieved that FGF2, activin-A, TGFβ, and their associatedreceptors represent the well-characterized signaling path-ways that maintain the undifferentiated states of epithelialhESCs (Fig. 2) (James et al., 2005; Ludwig et al., 2006). TGFβmaintains the pluripotent state of hESCs via receptor bindingto ALK5, which phosphorylates Smad2/3 and mediates thenuclear localization of the Smad2/3/4 complex on the targetgenes (James et al., 2005). Indeed, TGFβ alone was able tosustain the expression of hESC markers in TeSR1 medium(Ludwig et al., 2006).

Nonetheless, TGFβ is also involved in the regulation ofmultiphase differentiation such as mesodermal induction,myoblast proliferation, and invasion of cardiac jelly (Carlson,2009). It controls the EMT that drives gastrulation throughthe activation of Snail (Thiery et al., 2009), which thenincreases vimentin and concurrently decreases the expressionof epithelial genes such asDSP, KRT19, and the E-cad encodinggene CDH1. In addition, withdrawal of TGFβ from bovineserum and feeders enhances reprogramming to pluripotencyin mouse cells (Li et al., 2010), likely through the MET(Fig. 2C). TGFβ most likely modulates transient EMT eventsthat do not inhibit the pluripotent state of hESCs, whereasirreversible EMTs may be the major factor that initiatesdifferentiation both in vitro and in vivo (Fig. 2). Takentogether, these studies highlight the complexity of TGFβ instem cell biology, suggesting differential roles of TGFβ inreprogramming somatic cells, maintaining pluripotency, andmediating lineage differentiation (Fig. 2C). A thorough under-standing of the mechanisms that control intercellular interac-tions would enable us to optimize growth conditions, therebyminimizing cellular heterogeneity and abolishing potentialchromosomal abnormalities in hPSC culture.

616 K.G. Chen et al.

Chromosomal instability is still a problem inhuman pluripotent stem cell culture

During the development of early human embryos, chromosomeinstability (e.g., aneuploidies, uniparental disomies, segmentaldeletions, and amplifications) is a common feature in humancleavage-stage embryonic cells (Vanneste et al., 2009). Thus,the chromosomes of hPSCs and their derivatives might besimilarly altered at any stage during in vitro cell processing.

Although most lines were reported to be cytogeneticallynormal, there was a substantial number of cell lines thatacquire chromosomal changes after long-term culture, partic-ularly on chromosomes 1, 12, 17 and 20 (reviewed in Baker etal., 2007; Lee et al., 2013). Recently, Benvenisty and colleaguesanalyzed chromosomal changes in 104 hPSC lines, including 66hiPSC and 38 hESC samples from 18 different studies. Theyidentified ~20% abnormal lines, of which ~9% of the lines had atleast one trisomy (Mayshar et al., 2010). In a second cohortstudy, Andrews and colleagues reported 34% abnormal karyo-types in 129 hPSC lines, in which 20% of cytogenetically normallines carried a conserved amplicon at 20q11.12 (Amps et al.,2011). These studies define a putative rate of chromosomalabnormalities, ranging from 20% to 54%. The estimated rates forgenomic changes could be even higher if we could includeundefined small genomic changes such as point mutations.

It is believed that prolonged culture in vitro triggersselection of variant cells that have a higher tendency forsurvival and self-renewal (Baker et al., 2007; Lee et al.,2013). Notably, the methods employed for hPSC passagingmay have a significant impact on chromosomal changes. Ithas been shown that hPSCs with enzymatic mass-passaginghave a significant increase (i.e., 2-fold) in chromosomalabnormalities when compared with those cells using a manualcut-and-paste passaging technique (P = 0.017) (Amps et al.,2011). Nevertheless, the molecular basis that leads tochromosomal instability in hPSCs remains unclear. Recentstudies have linked chromosomal abnormalities to impairedDNA damage response and cell cycle checkpoint control(e.g., failure to activate CHK1 in hPSCs) (Desmarais et al.,2012; Hyka-Nouspikel et al., 2012). These studies highlight apotential important protective mechanism that hPSCs main-tain genomic stability by eradicating damaged cells throughapoptosis, rather than DNA repair or DNA homologousrecombination. Not surprisingly, Andrews and colleagueswere able to map the anti-apoptotic gene BCL2L1 on theconserved amplicon at 20q11.12, which may confer enhancedantiapoptotic activity in hPSCs with a normal karyotype (Ampset al., 2011).

Genomic changes at the chromosomal or single nucleotidelevel likely contribute to the variability of the pluripotentstates and differentiation potential. These changes can beacquired and accumulated during cell culture because ofsuboptimal growth media, inappropriate extracellular ma-trices, and dysregulated cell signals (Baker et al., 2007).With regard to hPSC culture, our goal is to avoid or minimizethe heterogeneity and the occurrence of these potentiallyharmful alterations that may possess tumorigenic effects.Notably, the chromosomal instability rates observed in hPSCare predominantly derived from prolonged hPSC culture ascolonies on MEFs and Matrigel. To improve upon colony-type-based culture methods, considerable efforts have been

made to develop alternative culture platforms for efficienthPSC expansion, differentiation, and high-throughput assays.

Robust growth models for pluripotent stem cellgrowth and assay

The need for clinically relevant quantities of hPSCs usedfor stem cell therapies and pharmaceutical applicationspromotes the development of compatible hPSC growthplatforms. Stem-cell based therapies usually demanddesired cell numbers ranging from 107 to 1010 or beyond.For example, repair of a 20-mm bone defect requiresabout 2 × 107 osteoblasts. Approximately 2 × 109 differenti-ated cardiomyocytes or insulin-producing beta cells areneeded to replace the damaged tissues after myocardialinfarction or for islet transplantation (Kehoe et al., 2010).The development of suspension culture and single-cellbased growth protocols for manufacturing hPSCs is an activearea of research. We have recently presented a comprehensiveanalysis of different culture protocols (Chen et al., 2014). In thefollowing sections, we will focus on several new methods basedon the variability, heterogeneity, chromosomal instability,and potential applications. These methods include suspen-sion culture of undifferentiated cells, single-cell passaging,and non-colony type monolayer (NCM).

Suspension culture in stirred-suspension bioreactors

Current suspension culture methods require that hESCcolonies are dissociated into small clumps to maintain theviability and pluripotency of the cells. To overcome the sizedifferences and variability of cell seeding, several groupshave adapted their methods from seeding small aggregatesto single-cells in stirred-suspension vessels (V.C. Chen etal., 2012; Kehoe et al., 2010; Krawetz et al., 2010; O'Brienand Laslett, 2012; Steiner et al., 2010). Typically, singlecells dissociated from hPSC colonies are incubated withMEF-conditioned medium containing 10% Matrigel and10 μM Y-27632 for 30 min to facilitate initial intercellularaggregation and introduced into spinner flasks with anagitation rate at 60 rpm for approximately 6 days followedby passaging (Kehoe et al., 2010). The suspension cell clusterswere passaged weekly by mechanically triturating the cellsinto smaller aggregates in the presence of Y-27632. Theseinvestigators believed that the suspended “EB-like” clusters ofhESCs maintained their pluripotency and did not turn intodifferentiated EBs (Steiner et al., 2010).

Benefits, pitfalls, and challenges of suspension cultureThe benefits of culturing hPSCs by stirred-suspensionbioreactors could be substantial: (i) rational scalability,(ii) reasonable hPSC expansion rates, (ii) elimination of feederand matrices, (iv) long-term maintenance of pluripotency(over 20 passages), and (v) compatibility with several formatssuch as microcarriers and microencapsulation (Chen et al.,2014). Not surprisingly, Chen and colleagues demonstratedthat they could maintain the pluripotency of hESCs andexpanded H9 (WA09) cells over 20 passages in suspensionculture, with a total fold expansion over 1 × 1013 (V.C.Chen et al., 2012). This represents approximately 4.3-fold

617Pluripotent stem cell growth models

expansion per passage in 3–4 days, which is much better thanadherent colonies (K.G. Chen et al., 2012). Encouragingly, thekaryotype appears to be normal after long-term suspensionculture in at least 10 different laboratories. Large cohortanalyses of chromosomal stability at a higher resolutionare necessary to ensure its superiority to other culturemethods. Nonetheless, the current methods still have severalpitfalls that need to be overcome prior to clinical applications,which include: (i) reduced cellular viability in bioreactorsdue to agitation-induced shear, (ii) dependence on the con-stant use of Y-27632, which induces aggregation, to improvecell survival rates, and (iii) increased differentiation andcellular heterogeneity.

Single-cell passaging and short-term assays

Single-cell passaging using ROCK inhibitors is now considered aroutine technique to promote single-cell survival (Watanabe etal., 2007). Ding and coworkers demonstrated that several novelsynthetic molecules could dramatically promote cloning effi-ciency of dissociated hESCs by enhancing ECM-mediated in-tegrin activity (Xu et al., 2010). Wu and colleagues assessed theeffects of small molecule combinations on colonies derivedfrom dissociated single cells and found several small moleculecocktails support long-term maintenance of hESCs (Tsutsui etal., 2011). This method can be used to dissociate hPSC coloniesto perform single-cells assays, cryopreservation, and gene-tic manipulation. However, the majority of studies usingsingle-cell passaging techniques employed low-density cellseeding and hence produced colonies. In addition, long-termsingle cell culture may induce chromosomal abnormalities.

Non-colony type monolayer (NCM) culture

We and others have recently developed a culture methodbased on single-cell passaging and NCM growth (K.G. Chenet al., 2012; Kunova et al., 2013). The key element inthis technology is high-density single-cell seeding, therebypermitting uniform multicellular association and avoiding theformation of colonies (Fig. 2B). There are many potentialadvantages to applying NCM for hPSC biology. An immediatebenefit of the NCM method is to accurately control growthrates, which yield approximately 3–4 fold average cell ex-pansion per passage in 3–4 days, comparable to the outcomesfrom some suspension cultures (Chen et al., 2014). The de-monstrated scalability of NCM culture to different sizes ofculture dishes may ensure hPSC expansion using currentlycompatible cell culture systems to produce clinically relevantcell quantities. Importantly, the NCM method aims to homo-genize heterogeneous cellular states in hPSC colonies. Indeed,cells under NCM conditions showed a more homogeneousresponse to signal molecules such as BMP-4 compared to thesame cells in colony-type culture (K.G. Chen et al., 2012).

In general, global gene expression patterns, determinedby cDNA microarray assays, were found to be similar toeach appropriate colony culture in three hPSC lines, withcorrelation ranges from 0.91 to 0.94. No EMT-related geneclusters were significantly increased in hPSCs under NCMconditions (K.G. Chen et al., 2012). Notably, the cell yieldsunder NCM conditions are better than colony-type culture,but less than suspension culture methods (Chen et al., 2014).

In our previous study, there were approximately 13% (2/16)lines showing chromosomal abnormalities (K.G. Chen et al.,2012). Since hPSCs under NCM conditions were adaptedfrom previous colony-type culture, it is unclear whetherthe chromosomal abnormalities observed in NCM culturewere due to the selection of preexisting mutant cells ofthe colony-type culture or generated de novo under NCM.Further comparative studies are needed to clarify this issue.

Current challenges and future directions

Obviously, colony type, non-colony type, and aggregatedcultures show many advantages in hPSC research. However,each method has its own limitations, which might undermineits capacity to produce clinical-grade hPSCs. In the nearfuture, we need to completely control the variability, hetero-geneity, and chromosomal instability to ensure faithful hPSCexpansion and differentiation. A systematic understanding ofthe mechanisms that contribute to the refractory properties ofhPSC culture would facilitate the achievement of these goals.Moreover, development of hierarchical growth models fororganogenesis would also have great clinical potential.

Control hPSC survival and fates with small molecules

The use of small molecules that inhibit or activate coresignaling pathways provides a powerful approach to supporthPSC survival and direct lineage commitment. However, thelong-term effects of small molecules on hPSC genomicintegrity and differentiation capacities have not been carefullyevaluated. For example, several culture protocols (as discussedabove) require the use of the ROCK inhibitor Y-27632 toenhance single-cell plating and stabilize intercellular aggrega-tions at every cell passage. Thus, the effects of Y-27632 onglobal gene expression and genomic stability should be carefullyevaluated. Under enzymatically dissociated conditions,damaged single cells are supposed to be eliminated byprogrammed cell death or apoptosis. It is unclear if ROCKinhibition-mediated single-cell survival also preserves apo-ptotic cells that might harbor potential genomic mutations.Verification of these mechanistic details would provide newstrategies to maintain genomic integrity of hPSCs.

Nevertheless, further modifications and characterizationof single-cell-based methods (such as suspension culture andNCM) are necessary to ensure its wide application for directeddifferentiation. First, to induce the cells to a specific precursorstage would facilitate directed differentiation of hPSCs undervarious culture conditions. It is likely that Y-27632-based NCMculture would enhance the differentiation efficiency of hPSCstoward neuroectodermal cells (K.G. Chen et al., 2012).We havealso tested the effects of various small molecules that interactwith ROCK signaling pathways on both single-cell platingand NCM growth. We found that Y-39983 (ROCK I inhibitor),phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (anovel ROCK inhibitor) can also modulate single-cell plating andpromote NCM growth. Theoretically, these small moleculesmaybe also used for suspension culture and single-cell based assays.Furthermore, high-throughput screening would enable us toidentify the next-generation of small molecules that drivedirected differentiation of hPSCs. Second, the development ofsingle-cell based methods without the use of small molecules,

618 K.G. Chen et al.

such as Y-27632, would be potentially important for cellexpansion and drug screening. The use of human recombinantlaminin-521 enables single-cell plating, thus permitting us toestablish NCM and suspension culture without the aid ofY-27632 and to produce homogeneous hPSCs in a chemicallydefined environment (Chen et al., unpublished data). Finally,it would be also possible to integrate optimized NCM andsuspension cultures into a well-controlled multidimensionalenvironment for tissue morphogenesis or organogenesis.

Figure 4 Intercellular interactions mediated by autocrine andmorphogenesis. (A) Stem cell niche maintenance exemplified by tVarious intercellular interactions are also depicted. (B) Modes of intand self-organization (morphogenesis). Abbreviations: hdFs, hESC-dIGF1R, insulin-like growth factor 1 receptor.

Modulation of intercellular interactionsfor organogenesis

Stem-cell niches, a hub for stem cell renewal and differen-tiation, are spatially oriented, temporally controlled, andusually heterogeneous micro-environments in mammaliantissues. ES cell fate determination can be traced in a 3Dstem cell niche (e.g., in hair-follicles) in animal models(Rompolas et al., 2013). It is unclear how hPSC niches are

paracrine signalings maintain stem cell niches and inducehe interaction between IGF-II (from hdFs) and IGF1R of hESCs.ercellular interactions induce hPSC differentiation, maturation,erived fibroblast-like cells; IGF-II, insulin-like growth factor 2;

619Pluripotent stem cell growth models

created in vitro microenvironment. Knowledge from bothdevelopmental biology and various hPSC culture models(Figs. 1–4) revealed that both autocrine and paracrinesignaling may play an important role in hPSC self-renewal,lineage differentiation, and tissue specification in an artificialstem cell niche.

Direct cell–cell interactions mediated byautocrine signalingThis type of interaction defines self-secretory ligandsbinding at corresponding receptors of the same cells or thesame types of cells (Fig. 4). These direct interactions maybe exemplified by Notch signaling which promotes neuraldifferentiation of hESCs. Activation of a highly conservedNotch pathway endorses neural lineage commitment in bothmESCs and hESCs (Lowell et al., 2006) and improves motorskills after ischemic injury in adult rat brain (Androutsellis-Theotokis et al., 2006). Stromal cells that express Notch ligandsalso encourage neural specification of hESCs. Hence, incom-plete activation of endogenous Notch results in heterogeneouslineage commitment. This neural commitment also necessi-tates a parallel signaling through the FGFR, highlighting thesignificance of cross-talk between core signaling pathways.

Indirect cell–cell interactions via paracrine signalingPluripotent stem cell fates are certainly influenced byextrinsic signals produced from heterogeneous types ofcells. Paracrine signaling from different types of cells mayhave differential effects on hPSC fates. It has been shownthat interplay between IGF-II (secreted from hESC-derivedfibroblast-like cells, i.e., hdFs) and IGF1R (constitutivelyexpressed in hESCs) promotes hESC self-renewal and thepluripotent state (Fig. 4A) (Bendall et al., 2007). Moreover,when hESCs were cocultured with human mesenchymal andumbilical vein endothelial cells, they formed self-organizedminiorganoids (i.e., liver buds) (Takebe et al., 2013). Thus,heterogeneous signaling appears to be required to inducelineage differentiation and subsequent tissue morphogene-sis (Fig. 4B). The generation of miniorganoids provides aproof-of-concept experiment that organogenesis can pro-ceed in a well-defined 3D environment. In the coming years,we anticipate using completely defined growth factors andsmall molecules to replace heterogeneous types of cells and togenerate pure populations of desired cells for transplantationtherapy in patients.

Conclusions

Human pluripotent stem cell growth models based on colony-type culture are widely used in stem cell research. However,these low efficient methods induce EMT, multicellularheterogeneity, and substantial genomic instability. EmerginghPSC growth models (such as NCM and suspension culture ofundifferentiated hPSCs) provide robust and scalable expansionof hPSCs as well as many other utilities, making them idealmethods to complement colony-type culture. With furtherunderstanding of core signaling pathways that controlintercellular interactions, efficient production of clinical-grade hPSCs and 3D-tissue morphogenesis in vitro couldbecome available for regenerative medicine and phar-maceutical development.

Disclosure of potential conflicts of interest

The authors indicate no potential conflict of interest.

Acknowledgments

This work was supported by the Intramural Research Programof the National Institutes of Health (NIH) at the NationalInstitute of Neurological Disorders and Stroke. We would liketo thank Dr. Peter Zandstra for his comments and suggestions.

References

Alikani, M., 2005. Epithelial cadherin distribution in abnormalhuman pre-implantation embryos. Hum. Reprod. 20, 3369–3375.

Amps, K., Andrews, P.W., Anyfantis, G., Armstrong, L., Avery, S.,Baharvand, H., Baker, J., Baker, D., Munoz, M.B., Beil, S., et al.,2011. Screening ethnically diverse human embryonic stem cellsidentifies a chromosome 20 minimal amplicon conferring growthadvantage. Nat. Biotechnol. 29, 1132–1144.

Androutsellis-Theotokis, A., Leker, R.R., Soldner, F., Hoeppner,D.J., Ravin, R., Poser, S.W., Rueger, M.A., Bae, S.K., Kittappa,R., McKay, R.D., 2006. Notch signalling regulates stem cellnumbers in vitro and in vivo. Nature 442, 823–826.

Baker, D.E., Harrison, N.J., Maltby, E., Smith, K., Moore, H.D., Shaw,P.J., Heath, P.R., Holden, H., Andrews, P.W., 2007. Adaptation toculture of human embryonic stem cells and oncogenesis in vivo.Nat. Biotechnol. 25, 207–215.

Bauwens, C.L., Peerani, R., Niebruegge, S., Woodhouse, K.A.,Kumacheva, E., Husain, M., Zandstra, P.W., 2008. Control ofhuman embryonic stem cell colony and aggregate size hetero-geneity influences differentiation trajectories. Stem Cells 26,2300–2310.

Behr, R., Heneweer, C., Viebahn, C., Denker, H.W., Thie, M., 2005.Epithelial–mesenchymal transition in colonies of rhesus monkeyembryonic stem cells: a model for processes involved in gastrula-tion. Stem Cells 23, 805–816.

Bendall, S.C., Stewart, M.H., Menendez, P., George, D., Vijayaragavan,K., Werbowetski-Ogilvie, T., Ramos-Mejia, V., Rouleau, A., Yang, J.,Bosse, M., et al., 2007. IGF and FGF cooperatively establish theregulatory stem cell niche of pluripotent human cells in vitro. Nature448, 1015–1021.

Braam, S.R., Zeinstra, L., Litjens, S., Ward-van Oostwaard, D., vanden Brink, S., van Laake, L., Lebrin, F., Kats, P., Hochstenbach, R.,Passier, R., et al., 2008. Recombinant vitronectin is a functionallydefined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells 26, 2257–2265.

Brafman, D.A., Phung, C., Kumar, N., Willert, K., 2012. Regulation ofendodermal differentiation of human embryonic stem cellsthrough integrin–ECM interactions. Cell Death Differ. 20, 369–381.

Bratt-Leal, A.M., Carpenedo, R.L., McDevitt, T.C., 2009. Engineeringthe embryoid body microenvironment to direct embryonic stemcell differentiation. Biotechnol. Prog. 25, 43–51.

Carlson, B.M. (Ed.), 2009. Human Embryology and DevelopmentalBiology, 4th edn. Mosby, Inc., Philadelphia.

Catalina, P., Montes, R., Ligero, G., Sanchez, L., de la Cueva, T.,Bueno, C., Leone, P.E., Menendez, P., 2008. Human ESCspredisposition to karyotypic instability: is a matter of cultureadaptation or differential vulnerability among hESC lines due toinherent properties? Mol. Cancer 7, 76.

Chen, K.G., Mallon, B.S., Hamilton, R.S., Kozhich, O.A., Park, K.,Hoeppner, D.J., Robey, P.G., McKay, R.D., 2012a. Non-colony typemonolayer culture of human embryonic stem cells. Stem Cell Res.9, 237–248.

620 K.G. Chen et al.

Chen, V.C., Couture, S.M., Ye, J., Lin, Z., Hua, G., Huang, H.I., Wu,J., Hsu, D., Carpenter, M.K., Couture, L.A., 2012b. Scalable GMPcompliant suspension culture system for human ES cells. Stem CellRes. 8, 388–402.

Chen, K.G., Mallon, B.S., McKay, R.D., Robey, P.G., 2014. Humanpluripotent stem cell culture: considerations for maintenance,expansion, and therapeutics. Cell Stem Cell 14, 13–26.

Dale, B., Gualtieri, R., Talevi, R., Tosti, E., Santella, L., Elder, K.,1991. Intercellular communication in the early human embryo.Mol. Reprod. Dev. 29, 22–28.

Dang, S.M., Gerecht-Nir, S., Chen, J., Itskovitz-Eldor, J., Zandstra,P.W., 2004. Controlled, scalable embryonic stem cell differentiationculture. Stem Cells 22, 275–282.

Davidson, K.C., Adams, A.M., Goodson, J.M., McDonald, C.E., Potter,J.C., Berndt, J.D., Biechele, T.L., Taylor, R.J., Moon, R.T., 2012.Wnt/beta-catenin signaling promotes differentiation, not self-renewal, of human embryonic stem cells and is repressed by Oct4.Proc. Natl. Acad. Sci. U. S. A. 109, 4485–4490.

Desmarais, J.A., Hoffmann, M.J., Bingham, G., Gagou, M.E., Meuth,M., Andrews, P.W., 2012. Human embryonic stem cells fail toactivate CHK1 and commit to apoptosis in response to DNAreplication stress. Stem Cells 30, 1385–1393.

Doble, B.W., Woodgett, J.R., 2007. Role of glycogen synthase kinase-3in cell fate and epithelial–mesenchymal transitions. Cells TissuesOrgans 185, 73–84.

Domogatskaya, A., Rodin, S., Boutaud, A., Tryggvason, K., 2008.Laminin-511 but not -332, -111, or -411 enables mouse embryonicstem cell self-renewal in vitro. Stem Cells 26, 2800–2809.

Domogatskaya, A., Rodin, S., Tryggvason, K., 2012. Functionaldiversity of laminins. Annu. Rev. Cell Dev. Biol. 28, 523–553.

Draper, J.S., Smith, K., Gokhale, P., Moore, H.D., Maltby, E.,Johnson, J., Meisner, L., Zwaka, T.P., Thomson, J.A., Andrews,P.W., 2004. Recurrent gain of chromosomes 17q and 12 incultured human embryonic stem cells. Nat. Biotechnol. 22,53–54.

Eastham, A.M., Spencer, H., Soncin, F., Ritson, S., Merry, C.L., Stern,P.L., Ward, C.M., 2007. Epithelial–mesenchymal transition eventsduring human embryonic stem cell differentiation. Cancer Res. 67,11254–11262.

Gualtieri, R., Santella, L., Dale, B., 1992. Tight junctions andcavitation in the human pre-embryo. Mol. Reprod. Dev. 32,81–87.

Gumbiner, B.M., 1996. Cell adhesion: the molecular basis of tissuearchitecture and morphogenesis. Cell 84, 345–357.

Hartung, O., Huo, H., Daley, G.Q., Schlaeger, T.M., 2010. Clumppassaging and expansion of human embryonic and inducedpluripotent stem cells on mouse embryonic fibroblast feedercells. Curr. Protoc. Stem Cell Biol. (Chapter 1, Unit 1C 10).

Hyka-Nouspikel, N., Desmarais, J., Gokhale, P.J., Jones, M., Meuth,M., Andrews, P.W., Nouspikel, T., 2012. Deficient DNA damageresponse and cell cycle checkpoints lead to accumulation ofpoint mutations in human embryonic stem cells. Stem Cells 30,1901–1910.

James, D., Levine, A.J., Besser, D., Hemmati-Brivanlou, A., 2005.TGFbeta/activin/nodal signaling is necessary for the maintenanceof pluripotency in human embryonic stem cells. Development 132,1273–1282.

Kehoe, D.E., Jing, D., Lock, L.T., Tzanakakis, E.S., 2010. Scalablestirred-suspension bioreactor culture of human pluripotent stemcells. Tissue Eng. Part A 16, 405–421.

Kleinman, H.K., McGarvey, M.L., Liotta, L.A., Robey, P.G.,Tryggvason, K., Martin, G.R., 1982. Isolation and characterizationof type IV procollagen, laminin, and heparan sulfate proteoglycanfrom the EHS sarcoma. Biochemistry 21, 6188–6193.

Krawetz, R., Taiani, J.T., Liu, S., Meng, G., Li, X., Kallos, M.S.,Rancourt, D.E., 2010. Large-scale expansion of pluripotent humanembryonic stem cells in stirred-suspension bioreactors. Tissue Eng.Part C Methods 16, 573–582.

Kunova, M., Matulka, K., Eiselleova, L., Salykin, A., Kubikova, I.,Kyrylenko, S., Hampl, A., Dvorak, P., 2013. Adaptation to robustmonolayer expansion produces human pluripotent stem cellswith improved viability. Stem Cells Transl. Med. 2, 246–254.

Lee, A.S., Tang, C., Rao, M.S., Weissman, I.L., Wu, J.C., 2013.Tumorigenicity as a clinical hurdle for pluripotent stem celltherapies. Nat. Med. 19, 998–1004.

Lefort, N., Feyeux, M., Bas, C., Feraud, O., Bennaceur-Griscelli,A., Tachdjian, G., Peschanski, M., Perrier, A.L., 2008. Humanembryonic stem cells reveal recurrent genomic instability at20q11.21. Nat. Biotechnol. 26, 1364–1366.

Li, X., Chen, Y., Scheele, S., Arman, E., Haffner-Krausz, R.,Ekblom, P., Lonai, P., 2001. Fibroblast growth factor signalingand basement membrane assembly are connected duringepithelial morphogenesis of the embryoid body. J. Cell Biol.153, 811–822.

Li, R., Liang, J., Ni, S., Zhou, T., Qing, X., Li, H., He, W., Chen, J., Li,F., Zhuang, Q., et al., 2010. Amesenchymal-to-epithelial transitioninitiates and is required for the nuclear reprogramming of mousefibroblasts. Cell Stem Cell 7, 51–63.

Lowell, S., Benchoua, A., Heavey, B., Smith, A.G., 2006. Notchpromotes neural lineage entry by pluripotent embryonic stemcells. PLoS Biol. 4, e121.

Ludwig, T.E., Levenstein, M.E., Jones, J.M., Berggren, W.T.,Mitchen, E.R., Frane, J.L., Crandall, L.J., Daigh, C.A., Conard, K.R.,Piekarczyk, M.S., et al., 2006. Derivation of human embryonic stemcells in defined conditions. Nat. Biotechnol. 24, 185–187.

Magyar, J.P., Nemir, M., Ehler, E., Suter, N., Perriard, J.C.,Eppenberger, H.M., 2001. Mass production of embryoid bodiesin microbeads. Ann. N. Y. Acad. Sci. 944, 135–143.

Mallon, B.S., Park, K.Y., Chen, K.G., Hamilton, R.S., McKay, R.D.,2006. Toward xeno-free culture of human embryonic stem cells.Int. J. Biochem. Cell Biol. 38, 1063–1075.

Maurer, J., Nelson, B., Cecena, G., Bajpai, R., Mercola, M., Terskikh,A., Oshima, R.G., 2008. Contrasting expression of keratins inmouse and human embryonic stem cells. PLoS One 3, e3451.

Mayshar, Y., Ben-David, U., Lavon, N., Biancotti, J.C., Yakir, B.,Clark, A.T., Plath, K., Lowry, W.E., Benvenisty, N., 2010.Identification and classification of chromosomal aberrationsin human induced pluripotent stem cells. Cell Stem Cell 7,521–531.

Miner, J.H., Li, C., Mudd, J.L., Go, G., Sutherland, A.E., 2004.Compositional and structural requirements for laminin andbasement membranes during mouse embryo implantation andgastrulation. Development 131, 2247–2256.

Moogk, D., Stewart, M., Gamble, D., Bhatia, M., Jervis, E., 2010.Human ESC colony formation is dependent on interplay betweenself-renewing hESCs and unique precursors responsible for nichegeneration. Cytometry A 77, 321–327.

O'Brien, C., Laslett, A.L., 2012. Suspended in culture—humanpluripotent cells for scalable technologies. Stem Cell Res. 9,167–170.

Padmanabhan, R., Chen, K.G., Gillet, J.P., Handley, M., Mallon, B.S.,Hamilton, R.S., Park, K., Varma, S., Mehaffey, M.G., Robey, P.G.,et al., 2012. Regulation and expression of the ATP-binding cassettetransporter ABCG2 in human embryonic stem cells. Stem Cells 30,2175–2187.

Pratama, G., Vaghjiani, V., Tee, J.Y., Liu, Y.H., Chan, J., Tan, C.,Murthi, P., Gargett, C., Manuelpillai, U., 2011. Changes in cultureexpanded human amniotic epithelial cells: implications forpotential therapeutic applications. PLoS One 6, e26136.

Rodin, S., Domogatskaya, A., Strom, S., Hansson, E.M., Chien, K.R.,Inzunza, J., Hovatta, O., Tryggvason, K., 2010. Long-termself-renewal of human pluripotent stem cells on humanrecombinant laminin-511. Nat. Biotechnol. 28, 611–615.

Rompolas, P., Mesa, K.R., Greco, V., 2013. Spatial organizationwithin a niche as a determinant of stem-cell fate. Nature 502,513–518.

621Pluripotent stem cell growth models

Sato, N., Meijer, L., Skaltsounis, L., Greengard, P., Brivanlou,A.H., 2004. Maintenance of pluripotency in human and mouseembryonic stem cells through activation of Wnt signaling by apharmacological GSK-3-specific inhibitor. Nat. Med. 10, 55–63.

Serra, M., Brito, C., Correia, C., Alves, P.M., 2012. Process engineeringof human pluripotent stem cells for clinical application. TrendsBiotechnol. 30, 350–359.

Steiner, D., Khaner, H., Cohen, M., Even-Ram, S., Gil, Y., Itsykson,P., Turetsky, T., Idelson, M., Aizenman, E., Ram, R., et al.,2010. Derivation, propagation and controlled differentiation ofhuman embryonic stem cells in suspension. Nat. Biotechnol. 28,361–364.

Stewart, M.H., Bosse, M., Chadwick, K., Menendez, P., Bendall, S.C.,Bhatia, M., 2006. Clonal isolation of hESCs reveals heterogeneitywithin the pluripotent stem cell compartment. Nat. Methods 3,807–815.

Takebe, T., Sekine, K., Enomura, M., Koike, H., Kimura, M., Ogaeri,T., Zhang, R.R., Ueno, Y., Zheng, Y.W., Koike, N., et al., 2013.Vascularized and functional human liver from an iPSC-derivedorgan bud transplant. Nature 499, 481–484.

Thiery, J.P., Acloque, H., Huang, R.Y., Nieto, M.A., 2009. Epithelial–mesenchymal transitions in development and disease. Cell 139,871–890.

Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A.,Swiergiel, J.J., Marshall, V.S., Jones, J.M., 1998. Embryonicstem cell lines derived from human blastocysts. Science 282,1145–1147.

Tsutsui, H., Valamehr, B., Hindoyan, A., Qiao, R., Ding, X., Guo, S.,Witte, O.N., Liu, X., Ho, C.M., Wu, H., 2011. An optimized smallmolecule inhibitor cocktail supports long-term maintenance ofhuman embryonic stem cells. Nat. Commun. 2, 167.

Twiss, F., de Rooij, J., 2013. Cadherin mechanotransduction in tissueremodeling. Cell. Mol. Life Sci. 70, 4101–4116.

Ullmann, U., In't Veld, P., Gilles, C., Sermon, K., De Rycke, M., Van deVelde, H., Van Steirteghem, A., Liebaers, I., 2007. Epithelial–mesenchymal transition process in human embryonic stem cellscultured in feeder-free conditions. Mol. Hum. Reprod. 13, 21–32.

Ullmann, U., Gilles, C., De Rycke, M., Van de Velde, H., Sermon, K.,Liebaers, I., 2008. GSK-3-specific inhibitor-supplemented hESCmedium prevents the epithelial–mesenchymal transition processand the up-regulation of matrix metalloproteinases in hESCscultured in feeder-free conditions. Mol. Hum. Reprod. 14, 169–179.

Ungrin, M.D., Joshi, C., Nica, A., Bauwens, C., Zandstra, P.W., 2008.Reproducible, ultra high-throughput formation of multicellularorganization from single cell suspension-derived human embryonicstem cell aggregates. PLoS One 3, e1565.

Vanneste, E., Voet, T., Le Caignec, C., Ampe, M., Konings, P.,Melotte, C., Debrock, S., Amyere, M., Vikkula, M., Schuit, F., et al.,2009. Chromosome instability is common in human cleavage-stageembryos. Nat. Med. 15, 577–583.

Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura,M., Wataya, T., Takahashi, J.B., Nishikawa, S., Nishikawa, S.,Muguruma, K., et al., 2007. A ROCK inhibitor permits survival ofdissociated human embryonic stem cells. Nat. Biotechnol. 25,681–686.

Xu, C., Inokuma, M.S., Denham, J., Golds, K., Kundu, P., Gold, J.D.,Carpenter, M.K., 2001. Feeder-free growth of undifferentiatedhuman embryonic stem cells. Nat. Biotechnol. 19, 971–974.

Xu, Y., Zhu, X., Hahm, H.S., Wei, W., Hao, E., Hayek, A., Ding, S.,2010. Revealing a core signaling regulatory mechanism forpluripotent stem cell survival and self-renewal by smallmolecules. Proc. Natl. Acad. Sci. U.S.A. 107, 8129–8134.