Applications of human embryonic stem cells (hESCs) are limited by the use of mouse embryonic fibroblasts feeder and animal-derived components during culture. In this study, we demonstrated the potential use of extracellular matrix (ECM) derived from the autologous feeders to support long-term undifferentiated growth of hESCs in xeno-free, serum-free, and feeder-free conditions. Autologous H9 ebF (feeder cells derived from outgrowth of embryoid body [EB] predifferentiated from H9 hESCs) was derived from EBs predifferentiated from H9 hESCs through a direct-plating outgrowth system. The ECM comprising collagen VI, laminin, and fibronectin was extracted from H9 ebF through a freeze–thaw procedure. The autologous ECM together with animal component-free TeSR™2 medium was used to support long-term growth of H1 and H9 hESC lines for up to 20 passages. The maintenance of hESC undifferentiated state by autologous ECM was confirmed by the positive staining of hESC-specific markers (Oct4, SSEA-4, and Tra-1-60) and the expression of pluripotency marker genes (Oct4, Nanog, and Sox2). Flow cytometry further showed that more than 99% of hESCs retained the expression of SSEA-3/4 after long-term culture on autologous ECM. Pluripotency of hESCs on ECM was further proven by in vitro EB formation and in vivo teratoma assay. Overall, this study suggested a strategy for efficient propagation of clinically compliant hESCs in an autologous feeder-free culture system.
Get full access to this article
View all access options for this article.
References
1.
ThomsonJ.A., Itskovitz-EldorJ., ShapiroS.S., WaknitzM.A., SwiergielJ.J., MarshallV.S.et al.Embryonic stem cell lines derived from human blastocysts. Science, 282:1145. 1998.
2.
AmitM., Itskovitz-EldorJ.Feeder-free culture of human embryonic stem cells. Methods Enzymol, 420:37. 2006.
3.
KleinmanH.K., McGarveyM.L., LiottaL.A., RobeyP.G., TryggvasonK., MartinG.R.Isolation and characterization of type IV procollagen, laminin, and heparan sulfate proteoglycan from the EHS sarcoma. Biochemistry, 21:6188. 1982.
4.
BissellD.M., ArensonD.M., MaherJ.J., RollF.J.Support of cultured hepatocytes by a laminin-rich gel. Evidence for a functionally significant subendothelial matrix in normal rat liver. J Clin Invest, 79:801. 1987.
5.
McGuireP.G., SeedsN.W.The interaction of plasminogen activator with a reconstituted basement membrane matrix and extracellular macromolecules produced by cultured epithelial cells. J Cell Biochem, 40:215. 1989.
6.
VukicevicS., KleinmanH.K., LuytenF.P., RobertsA.B., RocheN.S., ReddiA.H.Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp Cell Res, 202:1. 1992.
7.
FurueM.K., NaJ., JacksonJ.P., OkamotoT., JonesM., BakerD.et al.Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium. Proc Natl Acad Sci U S A, 105:13409. 2008.
XuR.H., PeckR.M., LiD.S., FengX., LudwigT., ThomsonJ.A.Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods, 2:185. 2005.
10.
LuJ., HouR., BoothC.J., YangS.H., SnyderM.Defined culture conditions of human embryonic stem cells. Proc Natl Acad Sci U S A, 103:5688. 2006.
11.
Sjogren-JanssonE., ZetterstromM., MoyaK., LindqvistJ., StrehlR., ErikssonP.S.Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system. Dev Dyn, 233:1304. 2005.
12.
VallierL., AlexanderM., PedersenR.A.Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci, 118:4495. 2005.
13.
YaoS., ChenS., ClarkJ., HaoE., BeattieG.M., HayekA.et al.Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions. Proc Natl Acad Sci U S A, 103:6907. 2006.
14.
LudwigT.E., LevensteinM.E., JonesJ.M., BerggrenW.T., MitchenE.R., FraneJ.L.et al.Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol, 24:185. 2006.
15.
WangL., SchulzT.C., SherrerE.S., DauphinD.S., ShinS., NelsonA.M.et al.Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood, 110:4111. 2007.
16.
KitajimaH., NiwaH.Clonal expansion of human pluripotent stem cells on gelatin-coated surface. Biochem Biophys Res Commun, 396:933. 2010.
AmitM., SharikiC., MarguletsV., Itskovitz-EldorJ.Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod, 70:837. 2004.
19.
AmitM., Itskovitz-EldorJ.Isolation and maintenance of primate ES cells. LanzaR.P.Handbook of Stem Cells2004 editionNew York: Elsevier Science, 2004; 1:419–436.
20.
BeattieG.M., LopezA.D., BucayN., HintonA., FirpoM.T., KingC.C.et al.Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells, 23:489. 2005.
21.
LiY., PowellS., BrunetteE., LebkowskiJ., MandalamR.Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products. Biotechnol Bioeng, 91:688. 2005.
22.
LiuY., SongZ., ZhaoY., QinH., CaiJ., ZhangH.et al.A novel chemical-defined medium with bFGF and N2B27 supplements supports undifferentiated growth in human embryonic stem cells. Biochem Biophys Res Commun, 346:131. 2006.
23.
AkopianV., AndrewsP.W., BeilS., BenvenistyN., BrehmJ., ChristieM.et al.Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells. In Vitro Cell Dev Biol Anim, 46:247. 2010.
24.
HakalaH., RajalaK., OjalaM., PanulaS., ArevaS., KellomakiM.et al.Comparison of biomaterials and extracellular matrices as a culture platform for multiple, independently derived human embryonic stem cell lines. Tissue Eng Part A, 15:1775. 2009.
25.
MooreH.The medium is the message. Nat Biotechnol, 24:160. 2006.
FuX., TohW.S., LiuH., LuK., LiM., HandeM.P.et al.Autologous feeder cells from embryoid body outgrowth support the long-term growth of human embryonic stem cells more effectively than those from direct differentiation. Tissue Eng Part C Methods, 16:719. 2010.
28.
Itskovitz-EldorJ., SchuldinerM., KarsentiD., EdenA., YanukaO., AmitM.et al.Differentiation of human embryonic stem cells into embryoid bodies compromising the three embryonic germ layers. Mol Med, 6:88. 2000.
29.
TohW.S., YangZ., LiuH., HengB.C., LeeE.H., CaoT.Effects of culture conditions and bone morphogenetic protein 2 on extent of chondrogenesis from human embryonic stem cells. Stem Cells, 25:950. 2007.
30.
FluckigerA.C., MarcyG., MarchandM., NegreD., CossetF.L., MitalipovS.et al.Cell cycle features of primate embryonic stem cells. Stem Cells, 24:547. 2006.
31.
YangZ., SuiL., TohW.S., LeeE.H., CaoT.Stage-dependent effect of TGF-beta1 on chondrogenic differentiation of human embryonic stem cells. Stem Cells Dev, 18:929. 2009.
32.
LivakK.J., SchmittgenT.D.Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods, 25:402. 2001.
33.
TohW.S., GuoX.M., ChooA.B., LuK., LeeE.H., CaoT.Differentiation and enrichment of expandable chondrogenic cells from human embryonic stem cells in vitro. J Cell Mol Med, 13:3570. 2009.
34.
SaylamC., OzdemirN., ItilI.M., SendagF., TerekM.C.Distribution of fibronectin, laminin and collagen type IV in the materno-fetal boundary zone of the developing mouse placenta. Experimental study. Arch Gynecol Obstet, 266:83. 2002.
35.
AlbertsB., JohnsonA., LewisJ., RaffM., RobertsK., WalterP.The extracellular matrix of animals. Molecular Biology of The Cell, 4th. New York: Garland Science, 2002; 1131–1195.
36.
AmentaP.S., GayS., VaheriA., Martinez-HernandezA.The extracellular matrix is an integrated unit: ultrastructural localization of collagen types I, III, IV, V, VI, fibronectin, and laminin in human term placenta. Coll Relat Res, 6:125. 1986.
37.
BeachamD.A., AmatangeloM.D., CukiermanE.Preparation of extracellular matrices produced by cultured and primary fibroblasts. Curr Protoc Cell BiolChapter 10, Unit 10.92007.
38.
NeganovaI., ZhangX., AtkinsonS., LakoM.Expression and functional analysis of G1 to S regulatory components reveals an important role for CDK2 in cell cycle regulation in human embryonic stem cells. Oncogene, 28:20. 2009.
39.
BeckerK.A., GhuleP.N., TherrienJ.A., LianJ.B., SteinJ.L., van WijnenA.J.et al.Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J Cell Physiol, 209:883. 2006.
40.
CukiermanE., PankovR., StevensD.R., YamadaK.M.Taking cell-matrix adhesions to the third dimension. Science, 294:1708. 2001.
41.
CoboF., StaceyG.N., HuntC., CabreraC., NietoA., MontesR.et al.Microbiological control in stem cell banks: approaches to standardisation. Appl Microbiol Biotechnol, 68:456. 2005.
RajalaK., LindroosB., HusseinS.M., LappalainenR.S., Pekkanen-MattilaM., InzunzaJ.et al.A defined and xeno-free culture method enabling the establishment of clinical-grade human embryonic, induced pluripotent and adipose stem cells. PLoS One, 5:e10246. 2010.
44.
SkottmanH., HovattaO.Culture conditions for human embryonic stem cells. Reproduction, 132:691. 2006.
45.
GuoX., HutcheonA.E., MelottiS.A., ZieskeJ.D., Trinkaus-RandallV., RubertiJ.W.Morphologic characterization of organized extracellular matrix deposition by ascorbic acid-stimulated human corneal fibroblasts. Invest Ophthalmol Vis Sci, 48:4050. 2007.
46.
SommerF., KobuchK., BrandlF., WildB., FrammeC., WeiserB.et al.Ascorbic acid modulates proliferation and extracellular matrix accumulation of hyalocytes. Tissue Eng, 13:1281. 2007.
47.
LareuR.R., SubramhanyaK.H., PengY., BennyP., ChenC., WangZ.et al.Collagen matrix deposition is dramatically enhanced in vitro when crowded with charged macromolecules: the biological relevance of the excluded volume effect. FEBS Lett, 581:2709. 2007.
48.
LareuR.R., ArsiantiI., SubramhanyaH.K., YanxianP., RaghunathM.In vitro enhancement of collagen matrix formation and crosslinking for applications in tissue engineering: a preliminary study. Tissue Eng, 13:385. 2007.
49.
HedmanK., KurkinenM., AlitaloK., VaheriA., JohanssonS., HookM.Isolation of the pericellular matrix of human fibroblast cultures. J Cell Biol, 81:83. 1979.
50.
PicacheE.R., HassaniehL., BroekD., SchonthalA.H.Inhibition of tumor cell growth by Triton X-100 through specific effects on cell-cycle-regulatory components. J Biomed Sci, 11:95. 2004.
51.
RotundaA.M., SuzukiH., MoyR.L., KolodneyM.S.Detergent effects of sodium deoxycholate are a major feature of an injectable phosphatidylcholine formulation used for localized fat dissolution. Dermatol Surg, 30:1001. 2004.
52.
PakzadM., TotonchiM., TaeiA., SeifinejadA., HassaniS.N., BaharvandH.Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging. Stem Cell Rev, 6:96. 2010.
53.
LiX., KrawetzR., LiuS., MengG., RancourtD.E.ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum Reprod, 24:580. 2009.
54.
BuenoC., MontesR., MenendezP.The ROCK inhibitor Y-27632 negatively affects the expansion/survival of both fresh and cryopreserved cord blood-derived CD34+ hematopoietic progenitor cells: Y-27632 negatively affects the expansion/survival of CD34+ HSPCs. Stem Cell Rev, 6:215. 2010.
55.
Di LulloG.A., SweeneyS.M., KorkkoJ., Ala-KokkoL., San AntonioJ.D.Mapping the ligand-binding sites and disease-associated mutations on the most abundant protein in the human, type I collagen. J Biol Chem, 277:4223. 2002.
56.
SteinH., WilenskyM., TsafrirY., RosenthalM., AmirR., AvrahamT.et al.Production of bioactive, post-translationally modified, heterotrimeric, human recombinant type-I collagen in transgenic tobacco. Biomacromolecules, 10:2640. 2009.
57.
RehnA.Regulation and function of mineralized tissue extra-cellular matrix proteins: studies of ADAMTS-1 and osteoadherin [Ph.D. thesis]Insistute of Odontology, Center for Oral Biology, Karolinska Institute, Stockholm, 2008.
58.
CormackD.H.Loose connective tissue and adipose tissue. CormackD.H.Essential Histology, 2nd. Philadeplhia: Lippincott Williams & Wilkins, 2001; 115–132.
59.
GarantP.R.Cytoskeleton, junctions, fibroblasts, and extracellular matrix. GarantP.R.Ten Cate's Oral Histology: Development, Structure, and Function, 7th. St. Louis, MO: Elsevier Health Sciences, 2007; 59–74.
60.
RoslerE.S., FiskG.J., AresX., IrvingJ., MiuraT., RaoM.S.et al.Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn, 229:259. 2004.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
