Restricted accessResearch articleFirst published online 2010-08
Autologous Feeder Cells from Embryoid Body Outgrowth Support the Long-Term Growth of Human Embryonic Stem Cells More Effectively than Those from Direct Differentiation
Autologous feeder cells have been developed by various methods to minimize the presence of xenogenic entities in human embryonic stem cell (hESC) cultures. However, there was no systematic comparison of supportive effects of the feeder cells on hESC growth, nor comparison to the supportive effects of various feeder-free culture systems and standard mouse feeder cells. In this study, we aimed to compare the supportive abilities of autologous feeders derived either directly from H9 hESCs (H9 dF) or from outgrowth of embryoid body predifferentiated in suspension from H9 hESCs (H9 ebF). Mouse feeder system and matrigel-mTeSR1 feeder-free system were used as controls. H9 ebF was found to secrete more basic fibroblast growth factor in the conditioned medium than H9 dF did. The undifferentiated state of H9 hESCs was sustained more stably on H9 ebF than on H9 dF, and the differentiation potential of H9 hESCs on H9 ebF was higher than on H9 dF. We concluded that H9 ebF was an optimal autologous feeder to maintain the long-term undifferentiated state of hESCs in our current culture system. This study helps to standardize the autologous culture of hESCs. It also suggests a more definite direction for future development of xeno-free culture system for hESCs.
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References
1.
ThomsonJ.A., Itskovitz-EldorJ., ShapiroS.S., WaknitzM.A., SwiergielJ.J., MarshallV.S., JonesJ.M.Embryonic stem cell lines derived from human blastocysts. Science, 282:1145. 1998.
2.
Gerecht-NirS., Itskovitz-EldorJ.Human embryonic stem cells: a potential source for cellular therapy. Am J Transplant, 4,Suppl 6:51. 2004.
3.
RoyA., KrzykwaE., LemieuxR., NéronS.Increased efficiency of gamma-irradiated versus mitomycin C-treated feeder cells for the expansion of normal human cells in long-term cultures. J Hematother Stem Cell Res, 10:873. 2001.
4.
XuC., InokumaM.S., DenhamJ., GoldsK., KunduP., GoldJ.D., CarpenterM.K.Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol, 19:971. 2001.
5.
Sjögren-JanssonE., ZetterströmM., 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.
6.
LudwigT.E., BergendahlV., LevensteinM.E., YuJ., ProbascoM.D., ThomsonJ.A.Feeder-independent culture of human embryonic stem cells. Nat Methods, 3:637. 2006.
7.
AmitM., SharikiC., MarguletsV., Itskovitz-EldorJ.Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod, 70:837. 2004.
8.
BendallS.C., StewartM.H., MenendezP., GeorgeD., VijayaragavanK., Werbowetski-OgilvieT., Ramos-MejiaV., RouleauA., YangJ., BosséM., LajoieG., BhatiaM.IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature, 448:1015. 2007.
9.
LevensteinM.E., LudwigT.E., XuR.H., LlanasR.A., VanDenHeuvel-KramerK., ManningD., ThomsonJ.A.Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells, 24:568. 2006.
10.
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.
11.
DvorakP., DvorakovaD., KoskovaS., VodinskaM., NajvirtovaM., KrekacD., HamplA.Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells. Stem Cells, 23:1200. 2005.
CoboF., NavarroJ.M., HerreraM.I., VivoA., PorcelD., HernándezC., JuradoM., García-CastroJ., MenendezP.Electron microscopy reveals the presence of viruses in mouse embryonic fibroblasts but neither in human embryonic fibroblasts nor in human mesenchymal cells used for hESC maintenance: toward an implementation of microbiological quality assurance program in stem cell banks. Cloning Stem Cells, 10:65. 2008.
RichardsM., TanS., FongC.Y., BiswasA., ChanW.K., BongsoA.Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. Stem Cells, 21:546. 2003.
16.
RichardsM., FongC.Y., ChanW.K., WongP.C., BongsoA.Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol, 20:933. 2002.
17.
ChengL., HammondH., YeZ., ZhanX., DravidG.Human adult marrow cells support prolonged expansion of human embryonic stem cells in culture. Stem Cells, 21:131. 2003.
18.
AmitM., CarpenterM.K., InokumaM.S., ChiuC.P., HarrisC.P., WaknitzM.A., Itskovitz-EldorJ., ThomsonJ.A.Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol, 227:271. 2000.
19.
HovattaO., MikkolaM., GertowK., StrömbergA.M., InzunzaJ., HreinssonJ., RozellB., BlennowE., AndängM., Ahrlund-RichterL.A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum Reprod, 18:1404. 2003.
20.
InzunzaJ., GertowK., StrömbergM.A., MatilainenE., BlennowE., SkottmanH., WolbankS., Ahrlund-RichterL., HovattaO.Derivation of human embryonic stem cell lines in serum replacement medium using postnatal human fibroblasts as feeder cells. Stem Cells, 23:544. 2005.
21.
XuC., JiangJ., SottileV., McWhirJ., LebkowskiJ., CarpenterM.K.Immortalized fibroblast-like cells derived from human embryonic stem cells support undifferentiated cell growth. Stem Cells, 22:972. 2004.
22.
YooS.J., YoonB.S., KimJ.M., SongJ.M., RohS., YouS., YoonH.S.Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell-derived feeder cells. Exp Mol Med, 37:399. 2005.
23.
SaxenaS., HanwateM., DebK., SharmaV., ToteyS.FGF2 secreting human fibroblast feeder cells: a novel culture system for human embryonic stem cells. Mol Reprod Dev, 75:1523. 2008.
24.
ChenH.F., ChuangC.Y., ShiehY.K., ChangH.W., HoH.N., KuoH.C.Novel autogenic feeders derived from human embryonic stem cells (hESCs) support an undifferentiated state of hESCs in xeno-free culture conditions. Reprod Biol, 24:1114. 2009.
25.
Itskovitz-EldorJ., SchuldinerM., KarsentiD., EdenA., YanukaO., AmitM., SoreqH., BenvenistyN.Differentiation of human embryonic stem cells into embryoid bodies comprising the three embryonic germ layers. Mol Med, 6:88. 2000.
26.
BoydN.L., DharaS.K., RekayaR., GodbeyE.A., HasneenK., RaoR.R., WestF.D.III, GerweB.A., SticeS.L.BMP4 promotes formation of primitive vascular networks in human embryonic stem cell-derived embryoid bodies. Exp Biol Med (Maywood), 232:833. 2007.
27.
FeraudO., CaoY., VittetD.Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis. Lab Invest, 81:1669. 2001.
28.
RisauW., SariolaH., ZerwesH.G., SasseJ., EkblomP., KemlerR., DoetschmanT.Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development, 102:471. 1988.
29.
FluckigerA.C., MarcyG., MarchandM., NégreD., CossetF.L., MitalipovS., WolfD., SavatierP., DehayC.Cell cycle features of primate embryonic stem cells. Stem Cells, 24:547. 2006.
30.
LillieR.D.Histopathologic Technic and Practical Histochemistry. 3rd edition. New York: McGraw-Hill Book Co, 1965.
31.
HaegelH., DierichA., CeredigR.CD44 in differentiated embryonic stem cells: surface expression and transcripts encoding multiple variants. Dev Immunol, 3:239. 1994.
32.
Maintenance of Human Embryonic Stem Cells in mTeSR1, Technical Manual, Stem Cell Technologies Inc., Version 1.1.0, 72008.
33.
HengB.C., RichardsM., CaoT.Are stem cells inherently more prone to cryopreservation-induced apoptosis compared to ordinary somatic cells?Hum Reprod, 24:492. 2009.
34.
HengB.C., YeC.P., LiuH., TohW.S., RufaihahA.J., YangZ., BayB.H., GeZ.G., OuyangH.W., LeeE.H., CaoT.Loss of viability during freeze-thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to apoptosis rather than cellular necrosis. J Biomed Sci, 13:433. 2006.
35.
HengB.C., KuleshovaL.L., BestedS.M., LiuH., CaoT.The cryopreservation of human embryonic stem cells. Biotechnol Appl Biochem, 41Pt 297. 2005.
36.
MeissnerA., WernigM., JaenischR.Direct reprogramming of genetically unmodified fibroblasts into pluripotent stem cells. Nat Biotechnol, 25:1177. 2007.
37.
PereiraR.C., EconomidesA.N., CanalisE.Bone morphogenetic proteins induce gremlin, a protein that limits their activity in osteoblasts. Endocrinology, 141:4558. 2000.
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., SteinG.S.Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J Cell Physiol, 209:883. 2006.
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