Human embryoid bodies to hepatocyte-like clusters:Preparing for translation☆

Giuseppe Pettinato,Melissa T.Thompson,Robert A.Fisher

Department of Surgery,Beth Israel Deaconess Medical Center,Harvard Medical School,Boston,MA,USA

1.Introduction

In the United States,more than 40,000 patients die of end-stage liverdisease and liverfailure,and an additional 2000 patients suffer from acute fulminant hepatic failure.1The only available treatment for most of these patients is liver transplantation.Livers from deceased donors account for 96%of the transplanted liver pool and represent the most common source for transplantation in the Western world.However,the high demand of donor livers creates an imbalance in which it outstrips supply.Therefore, finding alternatives to solid liver transplantation is an important strategy to increase the number of transplants and improve patient outcomes.

Hepatocytes have been used widely in drug evaluation and liver disease studiesin vitroand for transplantation.Although the techniques for isolating human hepatocytes are well established,it is the shortage of human liver tissue that is the major supply problem that limits this cellular therapy.Moreover,hepatocytes cannot be maintained easily in culture for extended periods.Furthermore,hepatocytes have limited ability to expandin vitro,even when specific growth factors are given(e.g.,hepatocyte growth factor).2Hepatocytes are also dif ficult to cryopreserve,because they are highly sensitive to freeze-thaw injury.3Therefore,alternative sources of hepatocytes are being examined to solve the dilemma.

Human induced pluripotent stem cells(hiPSCs)represent a promising option in regenerative medicine,based on their pluripotency,high proliferative capacity,and absence of ethical controversy.hiPSCs can be generated byretro-engineering a patient's cells into a pluripotent state through the addition of various stemness factors.4-6Differentiation of hiPSCs into hepatocyte-like cells(HLCs)is a potential cell therapy strategy for liver failure,bioengineered livers,and pharmaceutical testing.7However,the translational potential of stem cell-derived HLCs is limited by their scalability,immature genotypes post-differentiation,and poor long-term function after transplantation.8-10Recent studies have shown the potential of HLCs that are generated from hiPSCs.11,12These cells acquire the ability to secrete human albumin and alpha-1-antitrypsin(A1AT),synthesize urea,and regulate cytochrome P450(CYP)enzymesin vitro.

This review will focus on the latest research on HLCs that are derived from hiPSCs and the promising technology of human embryoid bodies(EBs).The formation of human embryoid bodies(hEBs)is particularly important,because a fundamental characteristic of living organisms is their cellular organization in a threedimensional structure,allowing adequate polarization and tissue organization in de fined functional structures.

2.Embryoid body formation for three-dimensional culture

The dedifferentiation of hepatocytes into a two-dimensional cell culture(monolayerculture)is awell-described phenomenon that is accompanied by a reduction in hepatocyte function,such as its detoxi fication properties and decreased plasma protein production(i.e.,albumin).13Two-dimensional culture forces adherent cells to modify their cytoskeleton toward a flattened morphology.This change in morphology limits cell-cell and cell-matrix interactions,diminishing cell polarization and impairing signaling pathways that are needed for normal hepatocyte function.13Such alterations are especially pertinent to hepatocytes,which are polygonal and have a multi-polarized structure with at least two basolateral and two apical surfaces.14Maintaining liver parenchymal functionex vivois needed for fully functional hepatocytes for use in toxicology screens,13for primary hepatocyte transplantation into patients,and the creation of bioarti ficial liver devices.The concept of full inducible hepato-cellular function has to be taken into consideration when using hiPSCs to differentiate HLCsin vitro.

The adequate and controlled differentiation of hiPSCs into a specific cell lineage with high throughput is fundamental for therapeutic purposes,especially when a considerable amount of one or more specific cell populations is needed.A common technique for differentiating hiPSCs requires the production of hEBs.EBs consist of a three-dimensional cell aggregate that resembles the structure of a developing embryo.15EBs allow us to generate cells from all three germ layers,and their differentiation relies on the nature of the EBs,which is in fluenced by the media,16the number of cells that constitute the EBs,and their size.17-19For instance,small EBs cannot survive differentiation,whereas those that are too large can result in core necrosis.19

Based on recent advances,EBs can be obtained using several methods:(i)by spontaneous self-aggregation in non-adhesive wells/dishes under static conditions,20(ii)in a hanging drop,21or(iii)by agitation(rotary culture,rocked culture,bioreactor)22or through microcavities and agarose micromolds.23-26

Conventional techniques for EB formation that are based on mechanical dissection of colonies have generated colony-derived EBs with a heterogeneous population that have not been reproducible with regard to size and cell type.27To ensure that EBs from hiPSCs are developed in a synchronized manner,the use of singularized hiPSCs is optimal,allowing strict control of the cell seeding density in every EB to manage its dimensions and consistency.

Several bioreactors have been constructed to produce hEBs and differentiate them in a precise scalable manner.28,29Regardless of the bene fits of this technology,the transplantation of differentiated cells using bioreactors has not shown any advantages for tissue replacement.30When hiPSCs are dissociated,they are at greater risk of apoptosis thanwhen theyare maintained as part of a colony,decreasing the rate of hEB formation from individualized hiPSCs.31Small molecules,such as the Rho-associated kinase(ROCK)inhibitor Y-27632,ensures the viability of singularized hiPSCs,most likely by preventing anoikis or promoting cell-cell contact to facilitate their aggregation.31Although ROCK inhibitor(ROCK-i)promotes the aggregation of dissociated hiPSCs,this small molecule might preclude the use of differentiated hiPSCs in a clinical setting.32Another method of promoting aggregation in a suspension of single hiPSCs is centrifugation(i.e.,the spin EB method).33This technique can induce damage in the hiPSC and might be an obstacle to automated scalable production of hEBs.34

In order to avoid the use of ROCK-i and centrifugation,our group developed a new technology that has allowed us to produce uniform hEBs from dissociated hiPSCs by using an agarose micromold.24,25Through precise control of the cell seeding density,we obtained homogeneous and synchronized hEBs in a scalable manner.Starting from a homogeneous pool of EBs,it was possible to effect more synchronous differentiation,such that all EBs could respond similarly to various growth factors.

3.Differentiation strategies

Before being considered for clinical application,HLCs that are obtained from hiPSCs have to be compared with primary hepatocytes and have similar morphology and function.In the past 10 years,many differentiation protocols have been published on the generation of HLCs from hESCs and hiPSCs.11,35-40All of these studies have shown that the differentiation and culture homogeneity are dependent on several variables in the culture system,including how the hiPSCs are cultured(two-dimensional vs.threedimensional),the differentiation protocol,and the scalability of the resulting differentiated cells.The best way to ef ficiently differentiate HLCs using hESCs and hiPSCs is by recapitulatingin vitrothe proper signaling pathways that are observed inin vivoembryo development studies.

Liver development in the embryo follows three steps:the formation of the de finitive endoderm(DE),hepatoblast formation and proliferation,and the differentiation of hepatoblasts into mature,functional hepatocytes.Hepatoblasts are bi-potential stem cells that give rise to the main cell lineages of the liver:hepatocytes and biliary epithelial cells(cholangiocytes).41

A sequence of signalsin vivodrives this process,which ultimately leads to liver organogenesis.In particular,specification of the mesendoderm,from which the mesenchyme and endoderm arise,is propelled by the nodal,bone morphogenetic protein(BMP),and activin signaling pathways.42,43In conjunction with activin-A,the stimulation of other pathways has also been demonstrated to promote endoderm formation,including fibroblast growth factor(FGF)and Wnt signaling.44,45In certain protocols,low doses of serum are required for activin-A to facilitate endoderm differentiation.11,42,46

Additional signaling from the FGF and BMP families,specifically by BMP4,FGF2,and FGF4,leads to the differentiation of hepatoblasts.11,47,48Following the formation of the liver bud,the inferential signals of hepatocyte growth factor(HGF)and oncostatin drive hepatoblasts to differentiate into hepatocytes.49

Despite the sequential administration of growth factors that are involved in hepatogenesis to differentiate hiPSCs through various stages,no hepatic differentiation protocol has addressed inhibition of the Wnt pathway,which occurs duringin vivoliver development.12,35,50-53The effects of Wnt/β-catenin signaling on cell differentiation into specific lineages,including hepatocytes,is widespread during embryogenesis across species,54and its in fluence on liver embryogenesis is highly regulated over time.55,56In the first stages of liver development,β-catenin expression is elevated at E10-E12,declining after E16.57,58During hepatogenesis,Wnt pathway regulation occurs late in cell differentiation and,in association withβ-catenin,is fundamental in mediating the differentiation of liver progenitor cells(i.e.,hepatoblasts)into hepatocytes or cholangiocytes.When activated,the Wnt/β-catenin pathway propels hepatoblasts toward cholangiocytes but drives hepatoblasts toward hepatocytes when inhibited.57,58

By taking advantage of these properties of the Wnt/β-catenin pathway,it is possible to manipulate the fate-determining hepatobiliary stage during differentiation to increase the yield of either cell phenotype.Incorporating inhibitors of the Wnt/β-catenin pathway as part of a differentiation protocol allows one to regulate the balance of fate specification into hepatocytes versus cholangiocytes,thus increasing hepatocyte production.57-60

Our group has developed a hepatic differentiation protocol that uses two Wnt/β-catenin signaling inhibitors,Wnt inhibitory factor(WIF)-1 and dickkopf-1(DKK-1),which has enabled us to increase the yield of hepatocyte-like cell differentiation beyond that obtained with other protocols.11Compared with human primary hepatocytes,our differentiated HLCs had many of the properties of primary hepatocytes,such as polygonal morphology,by light microscopy.By enzyme-linked immunosorbent assay(ELISA),our differentiated HLCs secreted several important hepatic proteins into the medium after 48 h of incubation.The concentration of humanalbuminrangedbetween120and130ng/mlfor 5×105cells,corresponding to approximately 60%of albumin production by human primary hepatocytes(128 ng/ml vs.199 ng/ml,P＜0.0009).Alpha fetoprotein(AFP)and fibrinogen levels were also comparable with those in human primary hepatocytes(AFP:0.18 ng/ml vs.0.19 ng/ml,P=0.69; fibrinogen:0.062 vs.0.064,P=0.0015).

Our differentiated HLCs also had functional activities that were typical of mature primary hepatocytes,such as acetylated lowdensity lipoprotein(DiI-ac-LDL)uptake,indocyanine green(ICG;Cardiogreen)absorption and release after 6 h,glycogen storage by periodic acid-Schiff(PAS)staining,and cytoplasmic accumulation of neutral triglycerides and lipids by oil red staining;these results were also comparable with those in human primary hepatocytes.By P450-GloTM assay,our differentiated HLCs responded to specific inducers,based on the increase in the activities of three isoforms of cytochrome P450(CYP1A2,CYP3A4,and CYP2B6)after stimulation.This detoxi fication pro file is characteristic at lower level of induction than with the one observed in human primary hepatocytes(CYP3A4:67 vs.82,P=0.0232;CYP2B6:14 vs.98,P＜0.0001;CYP1A2:22 vs.98,P＜0.0001).11

Finally,when we transplanted our differentiated HLCs in a rat model of acute liver failure,the survival rate increased the animals that were treated with our clusters,and human albumin was detected in the rat serum.11

In addition to administering soluble factors,hESCs and hiPSCs can be differentiated into HLCs by forced expression of transcription factors that are fundamental for liver organogenesis.Beginning with the generation of hiPSCs by Yamanaka's group in 2007,several teams have been able to reprogram somatic and embryonic stem cells into HLCs,bypassing the pluripotent stem cell stage.

Huang et al.51were the first to create HLCs from fibroblasts,demonstrating that the transduction of mouse fibroblasts fromp19arf-/-mice with GATA4,HNF1α,and FoxA3 promoted the generation of hepatic-like cells that expressed hepatic markers and restored liver function following transplantation in a mouse model.That same year,Sekiya et al.61used a group of transcription factors-HNF4α,FoxA1,and FoxA2 or FoxA3-to reprogram MEF into HLCs and showed that the resulting cells improved the survival of animals 10 weeks after cell transplantation by 40%.Two other groups have recently reported the ef ficacy of transduction using other transcription factors.52,62,63In particular,Nakamori et al.52generated more mature human HLCs by overexpressing activating transcription factor 5(ATF5),CCAAT/enhancer-binding protein alpha(c/EBPα),and prospero homeobox protein 1(PROX1);transduction of these three transcription factors upregulated several hepatic markers,including drug metabolism enzymes and hepatic transporters.By forcing the expression of HNF1b and FoxA3 and using specific hepatic culture media,Yahoo et al.62enhanced the hepatic lineage fate in mESCs.This group also concluded that the exogenous expression of HNF4a during directed differentiation could be an appropriate method for studying the effects of overexpression on hepatic differentiation of mESCs.63

Hepatic differentiation protocols have relied primarily on the use of human pluripotent stem cells,such as hESCs and hiPSCs,but other cell types have been used to differentiate HLCs.Mesenchymal stem cells from various tissues,such as bone marrow,adipose tissue,skin,placenta,and umbilical cord,have been differentiated into HLCs that have similar features as mature primary hepatocytes.64-71Our group differentiated human bone marrow stem cells into HLCs using a four-step differentiation protocol;72generating MSC-derived HLCs that can reverse liver failure and improve survival versus control animals.On transplantation,these cells functionedin vivo,as noted by the production of human albumin in the rat serum.72Table 1 shows the main differentiation protocols that have been applied for creating HLCs using hESCs and hiPSCs.Table 2 lists the cell types that have been used as sources of hepatocytes and HLCs for potential cell therapy.

4.Two-dimensional vs.three-dimensional culture

The ability to differentiate phenotypically correct cells from any type of tissue depends on the cocktail of growth factors that is used in the protocol and on the type of culture technique that is applied in the differentiation.Although two-dimensional cultures are adequate for learning basic cell biology,cells that are cultured with these methods acquire a flattened con figuration and experience altered cell-cell and cell-environment interactions.This structure negatively affectscellpolarization and modi fiesimportant signaling pathways,altering stem cell pluripotency and differentiation.73

Traditional hepatic differentiation protocols that have relied on two-dimensional adherence culture have generated cell populations that do not possess all of the features of primary hepatocytes.12,46,74,75During liver organogenesis,the liver bud is a threedimensionalstructure in which multiple celltypesinteract.9,10,53,76-78Cell-cell junctions,particularly through E-cadherin,have a positive impact on hepatocyte maturation.79,80Primary hepatocytes and hiPSC-derived HLCs that are grown in threedimensional culture maintain their hepatic properties better than their two-dimensional culture counterparts.81-87Some studies on hepatic differentiation using hiPSCs have coupled two-dimensional culture in the first steps of differentiation with three-dimensional culture for the final maturation of differentiated HLCs.50,85,88-92

Between two-dimensional culture-based differentiation and three-dimensional culture-based differentiation using hiPSC-EBs,the latter has several advantages,including better capacity for high cell density,by eliminating the cell-cell contact inhibition and growth that are typical of two-dimensional cultures and promoting maturation of HLCs through cell-cell contact.93Differentiating cells in two-dimensional cultures might have the advantage of having them have immediate access to the growth factors in the medium.However,tissues in the developing embryo arise from inductive signaling,which is determined by a three-dimensional structure in which a gradient is established within the germ layers.Similarly,the characteristic three-dimensional structure of EBs might mimic the microenvironment of thein vivoembryo,which might be a favorable condition for recreating this gradient diffusion and the proper signaling for tissue differentiationin vivo.One drawback of EBs is the presence of a core necrosis due to the poor diffusion of nutrients and oxygen in the center of the clusters.19However,this necrosis depends on the technique by which the EBs are obtained and can be avoided with bioengineering technologies,24,26,94-96and the use of supportive cells(e.g.,endothelial cells)that can aid nutrient exchange and engraftment after transplantation.72,97Table 2 lists the major two-dimensional and three-dimensional techniques that are used for each cell type.

Table 1Summary of standard protocols for hESC and hiPSC HLC differentiation.

Table 2Major advantages and drawbacks of each cell type and its respective 2D and 3D culture system.

5.Co-culture methods and use of extracellular matrices

In order to follow the signaling pathways that are observed during liver organogenesis in hiPSCs in a hepatic differentiation culture,several studies have explored the use of co-cultures as supporting cells to improve hepatic specification.For example,Pal et al.98utilized conditioned media from a human hepatocellular carcinoma(HepG2)cell line as a differentiation strategy using hESCs,and generated a large number of HCLs that were used to study thein vitrohepatic effects of ethanol toxicity.Fibroblast cells from various sources(STO feeder cells,3T3 cells,and pluripotent stem cells(PSCs)-derived fibroblast-like cells)have been used in various studies as adjuvant cells to enhance hepatic differentiation of hESCs and hiPSCsin vitro.99Other cells that participate in liver embryogenesis,including endothelial cells,mesenchymal cells,Kupffer cells,and stellate cells,have been used incorporated into differentiation protocols to improve hepatic specification and maturation in HLCs.100-103

We used a human endothelial cell line that was interlaced with hiPSCs and coupled to a three-dimensional culture to improve HLC engraftment after transplantation.72Transplantation of hiPSC EBs that were mixed with endothelial cells resulted in human albumin production for more than 14 days in 80%of the transplanted animals compared to the,suggesting that homogeneous mixing of endothelial cells and hiPSCs sustains the function of transplanted cells.By immunostaining of liver and spleen sections for endothelial cells,human endothelial cells persisted in the spleen but not in the liver,indicating that the HLCs homed to the injured liver and that human endothelial cells remained in the spleen.72

Our group also performed co-cultures using a monolayer of human primary hepatocytes as supporting cells in a threedimensional culture during the differentiation of hiPSC-EBs into HLCs.This strategy resulted in increased function of CYP enzymes,such as CYP1A2 and CYP3A4,compared with HCLs without coculture(Fig.1).Co-culture was also performed using 0.4μm permeable Transwell membrane filters;these filters were placed above the primary cell monolayer,and the differentiated hiPSC-EBs HCLs were added to the filter inserts,physically separating the EBs and primary cells.The 0.4μm filters allowed paracrine signaling through the diffusion of cytokines and signaling molecules but prevented the migration of larger molecules,such as albumin.As a result,in hiPSC-derived HCLs,the activity of the two CYPs increased,even in the presence of the filters,suggesting that cellcell interactions between primary hepatocytes and differentiated cells is not required for the induction of certain biochemical functions(Fig.1).

Another approach for improving hiPSC hepatic differentiationin vitrois the use of extracellular matrices or scaffolds that mimic the architecture of thein vivostructure of the developing liver.Various publications have highlighted various arti ficial and natural matrices to advance HCL differentiation,such as collagen type I,vitrogen,matrigel,polyurethane foam,104fibronectin,laminin,105polyacrylamide,106hollow fibers,86,107poly-l-lactic acid plus polyglycolic acid,108Ultra-Web nano fibers,109alginate microbeads,110nano fibrillar cellulose,hyaluronan-gelatin hydrogels,111and recombinant E-cadherin substratum.112Kanninen et al.113,114used acellular matrix from HepaRG cells.

Decellularization is an advanced technology that uses scaffolds and extracellular matrices to create a liver system that can be used for drug screens and clinical applications,in which an entire liver is repopulated with primary hepatocytes,hepatocyte cell lines(hepG2),or differentiated hiPSC-derived HLCs.Decellularized livers or extracellular matrices from decellularized livers have been used as three-dimensional sources for the culture of primary hepatocytes,because they are bio-resorbable,can be managed conveniently,function as support for long-term liver function,and have host specific native liver structure.115

Transplantation of primary hepatocytes that have been cultured with a synthetichydrogelfrom tissue-specific extracellular matrices placed into rats post-lethal-hepatectomy,reverses liver failure and extends survival.116A non-destructive method for monitoring cell removal during rat liver decellularization was recently developed by Geerts et al.117When decellularizing rat livers,this group used a protocol in which traditional destructive techniques were quality-controlled,based on DNA,collagen,and glycosaminoglycan(GAG)content in the scaffolds by histology.Computed tomography and perfusate analysis were also used as alternative nondestructive decellularization monitoring methods.As a result,they developed a decellularization procedure that generates scaffolds with considerably more GAG,without affecting their cell removal ef ficiency.

Mazza et al.118have advanced decellularized liver technologies,using human liver as supportive architectural structure for engineered livers,recellularizing cubical portions of an entire decellularized human liver with human cell lines,such as hepatic stellate cells(LX2),hepatocellular carcinoma(Sk-Hep-1),and hepatoblastoma(HepG2).Their experiments demonstrated the biocompatibility of the liver scaffold cubes that were transplanted subcutaneously into immune-competent mice,resulting in the absence of foreign body responses.Although these new technologies have made great strides toward the obtainment of functional liver structures,they still use cancer-or animal-derived cells and are thus unsuitable for human therapeutic purposes.

6.Future directions

A scalable supply of human hepatocytes will revolutionize the treatment of liver diseases and pharmaceutical testing.Potential sources of hepatocytes include hepatic progenitors from fetal or adult liver,differentiated pluripotent stem or mesenchymal stem cells,and direct reprogramming fromadult cells.hiPSC research is a constantly evolving field that can sidestep ethical problems and immune barriers with its use of hESCs.Areas of interest and potential treatment using hiPSC includein vitrohepatocellular functional modeling,in vivotreatment of primary liver diseases,testing of new drugs for hepatotoxicity,tissue engineering of liver structure,and development of bio-arti ficial liver(BAL)devices.

There is still considerable work that needs to be done before hiPSCs can be used for patient treatment,including:(i)improving the effectiveness of hiPSC production and avoiding the use of viral integration;(ii)bypassing the use of animal proteins in supernatants for the culture of hiPSCs;(iii)improving differentiation protocols for betterand more cost-effective generation of more mature cell types with comparable functions as primary hepatocytes;(iv)developing faster differentiation protocols for the eventual use of a patient's own cells for emergent applications;and(v)eliminating undifferentiated cell types that might form tumorsin vivo.

The accurate preclinical evaluation of hiPSCs in a large-animal model will be necessary to ensure that a therapeutic model that uses hiPSC-derived cells is safe and ef ficient before being tested in humans.Yamanaka's group recently showed that the tissue from which hiPSCs are obtained does notaffect hepatic differentiation,119but these variations are dependent on the donor cells from which the hiPSCs are derived.120

Thereareno standardized approachesforde fining the morphology,phenotype,and functional characteristics of differentiated HLCs between methods.Establishing a validated set of morphological and functional parameters for the evaluation of differentiated HLCs will be a key quality control step for future scale-up of HLCs for human therapies.

Fig.1.Co-culture of differentiated hiPSC-HLCs with primary hepatocyte cells results in increased function of CYP1A2 and CYP3A4 after stimulation with chemical inducers.Co-culture with(F)and without the filter improved the activity of P450 in differentiated HLCs.N=3.

Author's contribution

G.P.and R.A.F.designed experiments.G.P.and M.T.performed the experiments.G.P.and R.A.F.prepared the manuscript with contributions by all authors.

Con flict of interest

The authors declare that they have no con flict of interest.

Acknowledgment

This work was supported by Irvin Grant F36678.

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