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Factors from Human Embryonic Stem Cell-derived Fibroblast-like Cells Promote Topology-dependent Hepatic Differentiation in Primate Embryonic and Induced Pluripotent Stem Cells*

Open AccessPublished:August 18, 2010DOI:https://doi.org/10.1074/jbc.M110.122093
      The future clinical use of embryonic stem cell (ESC)-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. Here we report that a high density of human ESC-derived fibroblast-like cells (hESdFs) supported the efficient generation of hepatocyte-like cells with functional and mature hepatic phenotypes from primate ESCs and human induced pluripotent stem cells. Molecular and immunocytochemistry analyses revealed that hESdFs caused a rapid loss of pluripotency and induced a sequential endoderm-to-hepatocyte differentiation in the central area of ESC colonies. Knockdown experiments demonstrated that pluripotent stem cells were directed toward endodermal and hepatic lineages by FGF2 and activin A secreted from hESdFs. Furthermore, we found that the central region of ESC colonies was essential for the hepatic endoderm-specific differentiation, because its removal caused a complete disruption of endodermal differentiation. In conclusion, we describe a novel in vitro differentiation model and show that hESdF-secreted factors act in concert with regional features of ESC colonies to induce robust hepatic endoderm differentiation in primate pluripotent stem cells.

      Introduction

      Patients with end stage and genetic liver diseases have been shown to benefit from liver cell replacement therapy (
      • Akhter J.
      • Johnson L.A.
      • Gunasegaram A.
      • Riordan S.M.
      • Morris D.L.
      ). However, one of the major factors currently limiting the utilization of cellular replacement in the general population with severe liver disease is the lack of consistent sources of transplantable hepatocytes. Ideally, a proliferative stem cell population with robust capacity to generate hepatic cells in vitro will overcome the shortage of transplantable hepatic cells. Over the past several years, pluripotent embryonic stem cells (ESCs)
      The abbreviations used are: ESC
      embryonic stem cell
      hESdF
      human ESC-derived fibroblast-like cell
      HLC
      hepatocyte-like cell
      iPSC
      induced pluripotent stem cell
      BMP
      bone morphogenic factor
      HGF
      hepatocyte growth factor
      MEF
      mouse embryonic fibroblast
      ICC
      immunocytochemistry
      QRT-PCR
      quantitative RT-PCR
      IVDS
      in vitro differentiation stage
      HBV
      hepatitis B virus
      hESdF-CM
      hESdF-conditioned medium
      EB
      embryoid body.
      have been successfully derived from nonhuman primates (
      • Thomson J.A.
      • Kalishman J.
      • Golos T.G.
      • Durning M.
      • Harris C.P.
      • Becker R.A.
      • Hearn J.P.
      ,
      • Mitalipov S.
      • Kuo H.C.
      • Byrne J.
      • Clepper L.
      • Meisner L.
      • Johnson J.
      • Zeier R.
      • Wolf D.
      ,
      • Suemori H.
      • Tada T.
      • Torii R.
      • Hosoi Y.
      • Kobayashi K.
      • Imahie H.
      • Kondo Y.
      • Iritani A.
      • Nakatsuji N.
      ) and human embryos (
      • Reubinoff B.E.
      • Pera M.F.
      • Fong C.Y.
      • Trounson A.
      • Bongso A.
      ,
      • Thomson J.A.
      • Itskovitz-Eldor J.
      • Shapiro S.S.
      • Waknitz M.A.
      • Swiergiel J.J.
      • Marshall V.S.
      • Jones J.M.
      ). The ability of these cells to proliferate indefinitely and to develop into virtually any cell or tissue type, including hepatocytes, makes them likely candidates for cell-based treatment of human diseases.
      Over the past few decades, studies in vertebrate models have described several signaling pathways critical for the embryonic development of hepatocytes (
      • Zaret K.S.
      ,
      • Zhao R.
      • Duncan S.A.
      ). For example, it has been shown that selective FGFs can substitute for the cardiac mesoderm to activate the expression of hepatic genes in the endoderm (
      • Jung J.
      • Zheng M.
      • Goldfarb M.
      • Zaret K.S.
      ). In addition to the FGF pathway, bone morphogenic factors (BMPs), which are highly expressed in the septum transversum mesenchyme, are capable of inducing early hepatic fate independently in mice (
      • Rossi J.M.
      • Dunn N.R.
      • Hogan B.L.
      • Zaret K.S.
      ). Subsequently, Wnt signaling plays an important role in enhancing the growth of the liver bud (
      • Zaret K.S.
      ,
      • Monga S.P.
      • Pediaditakis P.
      • Mule K.
      • Stolz D.B.
      • Michalopoulos G.K.
      ). Other inductive signals, such as activin (
      • Weber H.
      • Holewa B.
      • Jones E.A.
      • Ryffel G.U.
      ,
      • D'Amour K.A.
      • Agulnick A.D.
      • Eliazer S.
      • Kelly O.G.
      • Kroon E.
      • Baetge E.E.
      ) and Sonic hedgehog (
      • Gualdi R.
      • Bossard P.
      • Zheng M.
      • Hamada Y.
      • Coleman J.R.
      • Zaret K.S.
      ,
      • Deutsch G.
      • Jung J.
      • Zheng M.
      • Lóra J.
      • Zaret K.S.
      ), also contribute to endoderm or early liver development. Terminal maturation of hepatocytes requires additional signals, such as hepatocyte growth factor (HGF) (
      • Schmidt C.
      • Bladt F.
      • Goedecke S.
      • Brinkmann V.
      • Zschiesche W.
      • Sharpe M.
      • Gherardi E.
      • Birchmeier C.
      ,
      • Kamiya A.
      • Kinoshita T.
      • Miyajima A.
      ) and oncostatin M (
      • Kamiya A.
      • Kinoshita T.
      • Miyajima A.
      ,
      • Kamiya A.
      • Kinoshita T.
      • Ito Y.
      • Matsui T.
      • Morikawa Y.
      • Senba E.
      • Nakashima K.
      • Taga T.
      • Yoshida K.
      • Kishimoto T.
      • Miyajima A.
      ).
      The future clinical use of ESC-based hepatocyte replacement therapy depends on the development of an efficient procedure for differentiation of hepatocytes from ESCs. To achieve this goal, researchers have successfully exploited the molecular signals for liver development outlined above to direct the development of mouse (
      • Hamazaki T.
      • Iiboshi Y.
      • Oka M.
      • Papst P.J.
      • Meacham A.M.
      • Zon L.I.
      • Terada N.
      ,
      • Yamamoto H.
      • Quinn G.
      • Asari A.
      • Yamanokuchi H.
      • Teratani T.
      • Terada M.
      • Ochiya T.
      ,
      • Gouon-Evans V.
      • Boussemart L.
      • Gadue P.
      • Nierhoff D.
      • Koehler C.I.
      • Kubo A.
      • Shafritz D.A.
      • Keller G.
      ) and subsequently monkey (
      • Tsukada H.
      • Takada T.
      • Shiomi H.
      • Torii R.
      • Tani T.
      ,
      • Momose Y.
      • Matsunaga T.
      • Murai K.
      • Takezawa T.
      • Ohmori S.
      ) and human (
      • Cai J.
      • Zhao Y.
      • Liu Y.
      • Ye F.
      • Song Z.
      • Qin H.
      • Meng S.
      • Chen Y.
      • Zhou R.
      • Song X.
      • Guo Y.
      • Ding M.
      • Deng H.
      ,
      • Duan Y.
      • Catana A.
      • Meng Y.
      • Yamamoto N.
      • He S.
      • Gupta S.
      • Gambhir S.S.
      • Zern M.A.
      ,
      • Hay D.C.
      • Fletcher J.
      • Payne C.
      • Terrace J.D.
      • Gallagher R.C.
      • Snoeys J.
      • Black J.R.
      • Wojtacha D.
      • Samuel K.
      • Hannoun Z.
      • Pryde A.
      • Filippi C.
      • Currie I.S.
      • Forbes S.J.
      • Ross J.A.
      • Newsome P.N.
      • Iredale J.P.
      ,
      • Hay D.C.
      • Zhao D.
      • Fletcher J.
      • Hewitt Z.A.
      • McLean D.
      • Urruticoechea-Uriguen A.
      • Black J.R.
      • Elcombe C.
      • Ross J.A.
      • Wolf R.
      • Cui W.
      ,
      • Basma H.
      • Soto-Gutiérrez A.
      • Yannam G.R.
      • Liu L.
      • Ito R.
      • Yamamoto T.
      • Ellis E.
      • Carson S.D.
      • Sato S.
      • Chen Y.
      • Muirhead D.
      • Navarro-Alvarez N.
      • Wong R.J.
      • Roy-Chowdhury J.
      • Platt J.L.
      • Mercer D.F.
      • Miller J.D.
      • Strom S.C.
      • Kobayashi N.
      • Fox I.J.
      ,
      • Chiao E.
      • Elazar M.
      • Xing Y.
      • Xiong A.
      • Kmet M.
      • Millan M.T.
      • Glenn J.S.
      • Wong W.H.
      • Baker J.
      ,
      • Agarwal S.
      • Holton K.L.
      • Lanza R.
      ) ESCs into hepatocyte-like cells (HLCs). Additionally, a few studies have demonstrated that mouse or monkey ESCs can be coaxed to generate HLCs in the presence of murine hepatocytes (
      • Saito K.
      • Yoshikawa M.
      • Ouji Y.
      • Moriya K.
      • Nishiofuku M.
      • Ueda S.
      • Hayashi N.
      • Ishizaka S.
      • Fukui H.
      ,
      • Fukumitsu K.
      • Ishii T.
      • Yasuchika K.
      • Amagai Y.
      • Saitoh M.
      • Kawamoto T.
      • Kawase E.
      • Suemori H.
      • Nakatsuji N.
      • Ikai I.
      • Uemoto S.
      ) or human liver nonparenchymal cells (
      • Soto-Gutiérrez A.
      • Navarro-Alvarez N.
      • Zhao D.
      • Rivas-Carrillo J.D.
      • Lebkowski J.
      • Tanaka N.
      • Fox I.J.
      • Kobayashi N.
      ).
      Recent demonstrations of the generation of induced pluripotent stem cells (iPSCs) with defined transcription factors (
      • Takahashi K.
      • Tanabe K.
      • Ohnuki M.
      • Narita M.
      • Ichisaka T.
      • Tomoda K.
      • Yamanaka S.
      ,
      • Lowry W.E.
      • Richter L.
      • Yachechko R.
      • Pyle A.D.
      • Tchieu J.
      • Sridharan R.
      • Clark A.T.
      • Plath K.
      ) that allowed the derivation of patient- and disease-specific pluripotent stem cells (
      • Park I.H.
      • Arora N.
      • Huo H.
      • Maherali N.
      • Ahfeldt T.
      • Shimamura A.
      • Lensch M.W.
      • Cowan C.
      • Hochedlinger K.
      • Daley G.Q.
      ,
      • Saha K.
      • Jaenisch R.
      ) without using human embryos have heightened interest in the in vitro hepatic differentiation potential of iPSCs (
      • Song Z.
      • Cai J.
      • Liu Y.
      • Zhao D.
      • Yong J.
      • Duo S.
      • Song X.
      • Guo Y.
      • Zhao Y.
      • Qin H.
      • Yin X.
      • Wu C.
      • Che J.
      • Lu S.
      • Ding M.
      • Deng H.
      ,
      • Sullivan G.J.
      • Hay D.C.
      • Park I.H.
      • Fletcher J.
      • Hannoun Z.
      • Payne C.M.
      • Dalgetty D.
      • Black J.R.
      • Ross J.A.
      • Samuel K.
      • Wang G.
      • Daley G.Q.
      • Lee J.H.
      • Church G.M.
      • Forbes S.J.
      • Iredale J.P.
      • Wilmut I.
      ,
      • Si-Tayeb K.
      • Noto F.K.
      • Nagaoka M.
      • Li J.
      • Battle M.A.
      • Duris C.
      • North P.E.
      • Dalton S.
      • Duncan S.A.
      ). The demonstration of the hepatic differentiation capability of iPSCs suggests their possible application for in vitro personalized pharmacogenetics, toxicology, and metabolism studies and future in vivo transplantation trails.
      In this study, we explored the potential of our previously established human ESC-derived fibroblast-like cells (hESdFs) (
      • Chen H.F.
      • Chuang C.Y.
      • Shieh Y.K.
      • Chang H.W.
      • Ho H.N.
      • Kuo H.C.
      ) for derivation of hepatocytes from pluripotent stem cells. We found that exposure of monkey and human ESCs and human iPSCs to a high density of hESdFs induced robust hepatic endoderm differentiation in a regionally specific manner in ESC/iPSC colonies. Additionally, we elaborate the possible mechanisms responsible for such hepatic induction.

      DISCUSSION

      Several protocols (
      • Tsukada H.
      • Takada T.
      • Shiomi H.
      • Torii R.
      • Tani T.
      ,
      • Momose Y.
      • Matsunaga T.
      • Murai K.
      • Takezawa T.
      • Ohmori S.
      ,
      • Cai J.
      • Zhao Y.
      • Liu Y.
      • Ye F.
      • Song Z.
      • Qin H.
      • Meng S.
      • Chen Y.
      • Zhou R.
      • Song X.
      • Guo Y.
      • Ding M.
      • Deng H.
      ,
      • Duan Y.
      • Catana A.
      • Meng Y.
      • Yamamoto N.
      • He S.
      • Gupta S.
      • Gambhir S.S.
      • Zern M.A.
      ,
      • Hay D.C.
      • Fletcher J.
      • Payne C.
      • Terrace J.D.
      • Gallagher R.C.
      • Snoeys J.
      • Black J.R.
      • Wojtacha D.
      • Samuel K.
      • Hannoun Z.
      • Pryde A.
      • Filippi C.
      • Currie I.S.
      • Forbes S.J.
      • Ross J.A.
      • Newsome P.N.
      • Iredale J.P.
      ,
      • Hay D.C.
      • Zhao D.
      • Fletcher J.
      • Hewitt Z.A.
      • McLean D.
      • Urruticoechea-Uriguen A.
      • Black J.R.
      • Elcombe C.
      • Ross J.A.
      • Wolf R.
      • Cui W.
      ,
      • Basma H.
      • Soto-Gutiérrez A.
      • Yannam G.R.
      • Liu L.
      • Ito R.
      • Yamamoto T.
      • Ellis E.
      • Carson S.D.
      • Sato S.
      • Chen Y.
      • Muirhead D.
      • Navarro-Alvarez N.
      • Wong R.J.
      • Roy-Chowdhury J.
      • Platt J.L.
      • Mercer D.F.
      • Miller J.D.
      • Strom S.C.
      • Kobayashi N.
      • Fox I.J.
      ,
      • Chiao E.
      • Elazar M.
      • Xing Y.
      • Xiong A.
      • Kmet M.
      • Millan M.T.
      • Glenn J.S.
      • Wong W.H.
      • Baker J.
      ,
      • Agarwal S.
      • Holton K.L.
      • Lanza R.
      ) have been published that claim to effectively coax human or monkey ESCs to differentiate into hepatocytes. The majority of the protocols described involve an initial differentiation step of embryoid body (EB) formation (
      • Tsukada H.
      • Takada T.
      • Shiomi H.
      • Torii R.
      • Tani T.
      ,
      • Momose Y.
      • Matsunaga T.
      • Murai K.
      • Takezawa T.
      • Ohmori S.
      ,
      • Duan Y.
      • Catana A.
      • Meng Y.
      • Yamamoto N.
      • He S.
      • Gupta S.
      • Gambhir S.S.
      • Zern M.A.
      ,
      • Basma H.
      • Soto-Gutiérrez A.
      • Yannam G.R.
      • Liu L.
      • Ito R.
      • Yamamoto T.
      • Ellis E.
      • Carson S.D.
      • Sato S.
      • Chen Y.
      • Muirhead D.
      • Navarro-Alvarez N.
      • Wong R.J.
      • Roy-Chowdhury J.
      • Platt J.L.
      • Mercer D.F.
      • Miller J.D.
      • Strom S.C.
      • Kobayashi N.
      • Fox I.J.
      ,
      • Chiao E.
      • Elazar M.
      • Xing Y.
      • Xiong A.
      • Kmet M.
      • Millan M.T.
      • Glenn J.S.
      • Wong W.H.
      • Baker J.
      ), and others include direct differentiation of ESCs on MEF (
      • Cai J.
      • Zhao Y.
      • Liu Y.
      • Ye F.
      • Song Z.
      • Qin H.
      • Meng S.
      • Chen Y.
      • Zhou R.
      • Song X.
      • Guo Y.
      • Ding M.
      • Deng H.
      ,
      • Agarwal S.
      • Holton K.L.
      • Lanza R.
      ) or Matrigel-coated plates (
      • Hay D.C.
      • Fletcher J.
      • Payne C.
      • Terrace J.D.
      • Gallagher R.C.
      • Snoeys J.
      • Black J.R.
      • Wojtacha D.
      • Samuel K.
      • Hannoun Z.
      • Pryde A.
      • Filippi C.
      • Currie I.S.
      • Forbes S.J.
      • Ross J.A.
      • Newsome P.N.
      • Iredale J.P.
      ,
      • Hay D.C.
      • Zhao D.
      • Fletcher J.
      • Hewitt Z.A.
      • McLean D.
      • Urruticoechea-Uriguen A.
      • Black J.R.
      • Elcombe C.
      • Ross J.A.
      • Wolf R.
      • Cui W.
      ). Subsequent strategies mostly involve multiple culture steps with sequential supplements of small molecules and recombinant growth factors to the culture medium. In this study, we demonstrate that the hESdF co-culture system is at least as efficient for hepatocyte differentiation as the previously published protocols (
      • Tsukada H.
      • Takada T.
      • Shiomi H.
      • Torii R.
      • Tani T.
      ,
      • Momose Y.
      • Matsunaga T.
      • Murai K.
      • Takezawa T.
      • Ohmori S.
      ,
      • Cai J.
      • Zhao Y.
      • Liu Y.
      • Ye F.
      • Song Z.
      • Qin H.
      • Meng S.
      • Chen Y.
      • Zhou R.
      • Song X.
      • Guo Y.
      • Ding M.
      • Deng H.
      ,
      • Duan Y.
      • Catana A.
      • Meng Y.
      • Yamamoto N.
      • He S.
      • Gupta S.
      • Gambhir S.S.
      • Zern M.A.
      ,
      • Hay D.C.
      • Fletcher J.
      • Payne C.
      • Terrace J.D.
      • Gallagher R.C.
      • Snoeys J.
      • Black J.R.
      • Wojtacha D.
      • Samuel K.
      • Hannoun Z.
      • Pryde A.
      • Filippi C.
      • Currie I.S.
      • Forbes S.J.
      • Ross J.A.
      • Newsome P.N.
      • Iredale J.P.
      ,
      • Hay D.C.
      • Zhao D.
      • Fletcher J.
      • Hewitt Z.A.
      • McLean D.
      • Urruticoechea-Uriguen A.
      • Black J.R.
      • Elcombe C.
      • Ross J.A.
      • Wolf R.
      • Cui W.
      ,
      • Basma H.
      • Soto-Gutiérrez A.
      • Yannam G.R.
      • Liu L.
      • Ito R.
      • Yamamoto T.
      • Ellis E.
      • Carson S.D.
      • Sato S.
      • Chen Y.
      • Muirhead D.
      • Navarro-Alvarez N.
      • Wong R.J.
      • Roy-Chowdhury J.
      • Platt J.L.
      • Mercer D.F.
      • Miller J.D.
      • Strom S.C.
      • Kobayashi N.
      • Fox I.J.
      ,
      • Chiao E.
      • Elazar M.
      • Xing Y.
      • Xiong A.
      • Kmet M.
      • Millan M.T.
      • Glenn J.S.
      • Wong W.H.
      • Baker J.
      ,
      • Agarwal S.
      • Holton K.L.
      • Lanza R.
      ). EB formation has been the most popular method exploited to differentiate ESCs into many cell types including hepatocytes; however, the uncontrolled nature of EB differentiation has made this approach less likely to generate scalable specific cell types with satisfactory purity. Monolayer-adherent culture conditions, on the other hand, have been used successfully to direct primate ESC differentiation into various cell types including hepatocytes (
      • Hay D.C.
      • Fletcher J.
      • Payne C.
      • Terrace J.D.
      • Gallagher R.C.
      • Snoeys J.
      • Black J.R.
      • Wojtacha D.
      • Samuel K.
      • Hannoun Z.
      • Pryde A.
      • Filippi C.
      • Currie I.S.
      • Forbes S.J.
      • Ross J.A.
      • Newsome P.N.
      • Iredale J.P.
      ,
      • Hay D.C.
      • Zhao D.
      • Fletcher J.
      • Hewitt Z.A.
      • McLean D.
      • Urruticoechea-Uriguen A.
      • Black J.R.
      • Elcombe C.
      • Ross J.A.
      • Wolf R.
      • Cui W.
      ). In this study, the efficient hepatocyte differentiation induced by the hESdF co-culture system required ESC/iPSCs to be cultured as a monolayer adhering to the culture surface. The monolayer-adherent culture helped to maintain the structural integrity of the two-dimensional ESC colonies and therefore possibly provided a foundation for establishing an unknown feature in the central region of ESC/iPSC cell colonies that is susceptible to differentiation signal(s) secreted by hESdFs.
      We showed that FGF2 and activin A secreted by hESdFs played critical roles in mediating the hepatic endoderm differentiation in primate pluripotent stem cells. This finding is well supported by results of previous reports, indicating that activin A and FGF2 have positive effects in promoting ESCs to differentiate into endoderms (
      • Basma H.
      • Soto-Gutiérrez A.
      • Yannam G.R.
      • Liu L.
      • Ito R.
      • Yamamoto T.
      • Ellis E.
      • Carson S.D.
      • Sato S.
      • Chen Y.
      • Muirhead D.
      • Navarro-Alvarez N.
      • Wong R.J.
      • Roy-Chowdhury J.
      • Platt J.L.
      • Mercer D.F.
      • Miller J.D.
      • Strom S.C.
      • Kobayashi N.
      • Fox I.J.
      ,
      • Soto-Gutiérrez A.
      • Navarro-Alvarez N.
      • Zhao D.
      • Rivas-Carrillo J.D.
      • Lebkowski J.
      • Tanaka N.
      • Fox I.J.
      • Kobayashi N.
      ). Unlike most protocols involving supplementation of extraneous factors (
      • Basma H.
      • Soto-Gutiérrez A.
      • Yannam G.R.
      • Liu L.
      • Ito R.
      • Yamamoto T.
      • Ellis E.
      • Carson S.D.
      • Sato S.
      • Chen Y.
      • Muirhead D.
      • Navarro-Alvarez N.
      • Wong R.J.
      • Roy-Chowdhury J.
      • Platt J.L.
      • Mercer D.F.
      • Miller J.D.
      • Strom S.C.
      • Kobayashi N.
      • Fox I.J.
      ,
      • Soto-Gutiérrez A.
      • Navarro-Alvarez N.
      • Zhao D.
      • Rivas-Carrillo J.D.
      • Lebkowski J.
      • Tanaka N.
      • Fox I.J.
      • Kobayashi N.
      ), it is not possible to manipulate the expression level of the factors secreted by hESdFs along the course of differentiation.
      However, we still observed efficient hepatocyte differentiation with the presence of mature hepatocytes. Therefore, the persistent presence of factors such as activin A and FGF2, which are important for early stages of differentiation, may not affect the subsequent differentiation and maturation of hepatocytes. Additionally, other growth factors secreted by hESdFs (Fig. 5B), such as BMP4, EGF, and VEGF, which have been shown to be required for hepatic specification of mouse ESC-derived endoderm (
      • Gouon-Evans V.
      • Boussemart L.
      • Gadue P.
      • Nierhoff D.
      • Koehler C.I.
      • Kubo A.
      • Shafritz D.A.
      • Keller G.
      ,
      • Runge D.M.
      • Runge D.
      • Dorko K.
      • Pisarov L.A.
      • Leckel K.
      • Kostrubsky V.E.
      • Thomas D.
      • Strom S.C.
      • Michalopoulos G.K.
      ) and to enhance hepatocyte specification of ESCs, respectively, were very likely responsible for the hepatic specification, differentiation, and maturation following the endodermal differentiation mediated by hESdF secreted FGF2 and activin A (
      • Gouon-Evans V.
      • Boussemart L.
      • Gadue P.
      • Nierhoff D.
      • Koehler C.I.
      • Kubo A.
      • Shafritz D.A.
      • Keller G.
      ). Finally, the nonparenchymal cells generated along the hepatic lineages differentiation may also contribute to the differentiation and maturation process by secreting growth factors, because HGF expression was normally detected in hepatic foci after 3 weeks of differentiation (Fig. 5B). Taken together, these results strongly suggest that a combination of hESdF and differentiated ESC secreted factors might be responsible for the hepatocyte differentiation and maturation in our system.
      In the hESdF co-culture system, the central regions of ESC colonies always transformed into endoderm-like foci, a step that preceded hepatocyte differentiation. Removal of central foci disrupted the progress of hepatic endoderm differentiation and resulted in the acquisition of ectodermal fate by the regenerated cells. We can only speculate about why the central region of the ESC colonies appears so important for hepatic differentiation. It seems unlikely that the hESdF feeders created a growth factor/cytokine signal gradient along the central to peripheral axis of the ESC colonies, because the differentiation foci could still be readily induced in hESdF-conditioned medium alone.
      Alternatively, there might be heterogeneous populations of pluripotent cells in ESC colonies that are assembled in specific topological compartments in the two-dimensional ESC/iPSC colonies; perhaps only those allocated to the central region of ESC/iPSC colonies could properly respond to hESdF-secreted factors to form hepatic foci. This hypothesis is consistent with a recent observation showing that subpopulations of ESCs with distinct tissue-specific fates can be selected from pluripotent cultures (
      • King F.W.
      • Ritner C.
      • Liszewski W.
      • Kwan H.C.
      • Pedersen A.
      • Leavitt A.D.
      • Bernstein H.S.
      ) and was supported by our microarray analysis comparing the gene expression patterns of the central and rim regions of the ESC colonies (Fig. 3). Future identification of the molecular mechanism by which the distinct hepatocyte-promoting capability of the central region of ESC colonies is established may lead to more efficient control of hepatic differentiation.
      Consistent with a previous study (
      • Si-Tayeb K.
      • Noto F.K.
      • Nagaoka M.
      • Li J.
      • Battle M.A.
      • Duris C.
      • North P.E.
      • Dalton S.
      • Duncan S.A.
      ), we found that different iPSC lines have different capabilities to generate hepatic lineages in response to differentiation stimuli (e.g. hESdF treatment), even though that they are all regarded as fully reprogrammed iPSC lines according to currently used standards (supplemental Fig. S7) for iPSC characterization. These discrepancies may be attributed to currently unknown factors that modulate hepatocyte differentiation, which were somehow defective in some of the iPSC lines. It will be of interest to further explore the mechanisms that constitute the discrepancies among different iPSC clones.
      In summary, we have developed a simple method that allows scalable hepatocyte generation from primate pluripotent stem cells. Furthermore, we provide compelling evidence indicating that hESdF-secreted factors are responsible for the topology-dependent differentiation of endodermal cells to hepatocytes in ESC/iPSC colonies. These findings provide opportunities to further explore the mechanisms underpinning hepatic lineage differentiation and may be useful for future pharmacotoxicology and transplantation applications.

      Acknowledgments

      We thank Drs. Chun-Jen Liu and Yung-Ming Jeng for sera and liver sections, respectively, from HBV-infected patients. We thank the National RNAi Core Facility (supported by National Science Council Grant 97-3112-B-001-016) for lentiviral shRNA clones and the Affymetrix Gene Expression Service Lab of Academia Sinica for microarray assays.

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