Journal of Molecular Medicine

, Volume 86, Issue 8, pp 875–886 | Cite as

Deconstructing human embryonic stem cell cultures: niche regulation of self-renewal and pluripotency

  • Morag H. Stewart
  • Sean C. Bendall
  • Mickie Bhatia
Review

Abstract

The factors and signaling pathways controlling pluripotent human cell properties, both embryonic and induced, have not been fully investigated. Failure to account for functional heterogeneity within human embryonic stem cell (hESC) cultures has led to inconclusive results in previous work examining extrinsic influences governing hESC fate (self renewal vs. differentiation vs. death). Here, we attempt to reconcile these inconsistencies with recent reports demonstrating that an autologously produced in vitro niche regulates hESCs. Moreover, we focus on the reciprocal paracrine signals within the in vitro hESC niche allowing for the maintenance and/or expansion of the hESC colony-initiating cell (CIC). Based on this, it is clear that separation of hESC-CICs, apart from their differentiated derivatives, will be essential in future studies involving their molecular regulation. Understanding how extrinsic factors control hESC self-renewal and differentiation will allow us to culture and differentiate these pluripotent cells with higher efficiency. This knowledge will be essential for clinical applications using human pluripotent cells in regenerative medicine.

Keywords

Human embryonic stem cells Niche Differentiation Self-renewal Pluripotency Signaling 

References

  1. 1.
    Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147PubMedCrossRefGoogle Scholar
  2. 2.
    Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A (2000) Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat Biotechnol 18:399–404PubMedCrossRefGoogle Scholar
  3. 3.
    Amit M, Carpenter MK, Inokuma MS, Chiu CP, Harris CP, Waknitz MA, Itskovitz-Eldor J, Thomson JA (2000) Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev Biol 227:271–278PubMedCrossRefGoogle Scholar
  4. 4.
    Nakagawa M, Koyanagi M, Tanabe K, Takahashi K, Ichisaka T, Aoi T, Okita K, Mochiduki Y, Takizawa N, Yamanaka S (2008) Generation of induced pluripotent stem cells without Myc from mouse and human fibroblasts. Nat Biotechnol 26:101–106PubMedCrossRefGoogle Scholar
  5. 5.
    Park IH, Zhao R, West JA, Yabuuchi A, Huo H, Ince TA, Lerou PH, Lensch MW, Daley GQ (2008) Reprogramming of human somatic cells to pluripotency with defined factors. Nature 451:141–146PubMedCrossRefGoogle Scholar
  6. 6.
    Hanna J, Wernig M, Markoulaki S, Sun CW, Meissner A, Cassady JP, Beard C, Brambrink T, Wu LC, Townes TM, Jaenisch R (2007) Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318:1920–1923PubMedCrossRefGoogle Scholar
  7. 7.
    Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, Nie J, Jonsdottir GA, Ruotti V, Stewart R, Slukvin II, Thomson JA (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858):1917–1920PubMedCrossRefGoogle Scholar
  8. 8.
    Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861–872PubMedCrossRefGoogle Scholar
  9. 9.
    Chase LG, Firpo MT (2007) Development of serum-free culture systems for human embryonic stem cells. Curr Opin Chem Biol 11:367–372PubMedCrossRefGoogle Scholar
  10. 10.
    Lei T, Jacob S, Ajil-Zaraa I, Dubuisson JB, Irion O, Jaconi M, Feki A (2007) Xeno-free derivation and culture of human embryonic stem cells: current status, problems and challenges. Cell Res 17:682–688PubMedCrossRefGoogle Scholar
  11. 11.
    Skottman H, Narkilahti S, Hovatta O (2007) Challenges and approaches to the culture of pluripotent human embryonic stem cells. Regen Med 2:265–273PubMedCrossRefGoogle Scholar
  12. 12.
    Ludwig TE, Levenstein ME, Jones JM, Berggren WT, Mitchen ER, Frane JL, Crandall LJ, Daigh CA, Conard KR, Piekarczyk MS, Llanas RA, Thomson JA (2006) Derivation of human embryonic stem cells in defined conditions. Nat Biotechnol 24:185–187PubMedCrossRefGoogle Scholar
  13. 13.
    Draper JS, Smith K, Gokhale P, Moore HD, Maltby E, Johnson J, Meisner L, Zwaka TP, Thomson JA, Andrews PW (2004) Recurrent gain of chromosomes 17q and 12 in cultured human embryonic stem cells. Nat Biotechnol 22:53–54PubMedCrossRefGoogle Scholar
  14. 14.
    Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK (2001) Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 19:971–974PubMedCrossRefGoogle Scholar
  15. 15.
    Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R (2006) Human embryonic stem cell lines derived from single blastomeres. Nature 444(7118):481–485PubMedCrossRefGoogle Scholar
  16. 16.
    Bendall SC, Stewart MH, Menendez P, George D, Vijayaragavan K, Werbowetski-Ogilvie T, Ramos-Mejia V, Rouleau A, Yang J, Bosse M, Lajoie G, Bhatia M (2007) IGF and FGF cooperatively establish the regulatory stem cell niche of pluripotent human cells in vitro. Nature 448:1015–1021PubMedCrossRefGoogle Scholar
  17. 17.
    Stewart MH, Bosse M, Chadwick K, Menendez P, Bendall SC, Bhatia M (2006) Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment. Nat Methods 3:807–815PubMedCrossRefGoogle Scholar
  18. 18.
    Greber B, Lehrach H, Adjaye J (2007) Fibroblast growth factor 2 modulates transforming growth factor beta signaling in mouse embryonic fibroblasts and human ESCs (hESCs) to support hESC self-renewal. Stem Cells 25:455–464PubMedCrossRefGoogle Scholar
  19. 19.
    Scadden DT (2006) The stem-cell niche as an entity of action. Nature 441:1075–1079PubMedCrossRefGoogle Scholar
  20. 20.
    Jones DL, Wagers AJ (2008) No place like home: anatomy and function of the stem cell niche. Nat Rev Mol Cell Biol 9:11–21PubMedCrossRefGoogle Scholar
  21. 21.
    Li L, Xie T (2005) Stem cell niche: structure and function. Annu Rev Cell Dev Biol 21:605–631PubMedCrossRefGoogle Scholar
  22. 22.
    Schofield R (1978) The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 4:7–25PubMedGoogle Scholar
  23. 23.
    Kiger AA, White-Cooper H, Fuller MT (2000) Somatic support cells restrict germline stem cell self-renewal and promote differentiation. Nature 407:750–754PubMedCrossRefGoogle Scholar
  24. 24.
    Xie T, Spradling AC (2000) A niche maintaining germ line stem cells in the Drosophila ovary. Science 290:328–330PubMedCrossRefGoogle Scholar
  25. 25.
    Crittenden SL, Bernstein DS, Bachorik JL, Thompson BE, Gallegos M, Petcherski AG, Moulder G, Barstead R, Wickens M, Kimble J (2002) A conserved RNA-binding protein controls germline stem cells in Caenorhabditis elegans. Nature 417:660–663PubMedCrossRefGoogle Scholar
  26. 26.
    Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703–716PubMedCrossRefGoogle Scholar
  27. 27.
    Seri B, Garcia-Verdugo JM, McEwen BS, Alvarez-Buylla A (2001) Astrocytes give rise to new neurons in the adult mammalian hippocampus. J Neurosci 21:7153–7160PubMedGoogle Scholar
  28. 28.
    Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, Clevers H (2007) Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449:1003–1007PubMedCrossRefGoogle Scholar
  29. 29.
    Blanpain C, Horsley V, Fuchs E (2007) Epithelial stem cells: turning over new leaves. Cell 128:445–458PubMedCrossRefGoogle Scholar
  30. 30.
    Lee JB, Song JM, Lee JE, Park JH, Kim SJ, Kang SM, Kwon JN, Kim MK, Roh SI, Yoon HS (2004) Available human feeder cells for the maintenance of human embryonic stem cells. Reproduction 128:727–735PubMedCrossRefGoogle Scholar
  31. 31.
    Stojkovic P, Lako M, Stewart R, Przyborski S, Armstrong L, Evans J, Murdoch A, Strachan T, Stojkovic M (2005) An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23:306–314PubMedCrossRefGoogle Scholar
  32. 32.
    Yoo SJ, Yoon BS, Kim JM, Song JM, Roh S, You S, Yoon HS (2005) Efficient culture system for human embryonic stem cells using autologous human embryonic stem cell-derived feeder cells. Exp Mol Med 37:399–407PubMedGoogle Scholar
  33. 33.
    Prowse AB, McQuade LR, Bryant KJ, Marcal H, Gray PP (2007) Identification of potential pluripotency determinants for human embryonic stem cells following proteomic analysis of human and mouse fibroblast conditioned media. J Proteome Res 6:3796–3807PubMedCrossRefGoogle Scholar
  34. 34.
    Lim JW, Bodnar A (2002) Proteome analysis of conditioned medium from mouse embryonic fibroblast feeder layers which support the growth of human embryonic stem cells. Proteomics 2:1187–1203PubMedCrossRefGoogle Scholar
  35. 35.
    Prowse AB, McQuade LR, Bryant KJ, Van Dyk DD, Tuch BE, Gray PP (2005) A proteome analysis of conditioned media from human neonatal fibroblasts used in the maintenance of human embryonic stem cells. Proteomics 5(4):978–989PubMedCrossRefGoogle Scholar
  36. 36.
    Becker AJ, Mc CE, Till JE (1963) Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197:452–454PubMedCrossRefGoogle Scholar
  37. 37.
    Enver T, Soneji S, Joshi C, Brown J, Iborra F, Orntoft T, Thykjaer T, Maltby E, Smith K, Dawud RA, Jones M, Matin M, Gokhale P, Draper J, Andrews PW (2005) Cellular differentiation hierarchies in normal and culture-adapted human embryonic stem cells. Hum Mol Genet 14:3129–3140PubMedCrossRefGoogle Scholar
  38. 38.
    Mantel C, Guo Y, Lee MR, Kim MK, Han MK, Shibayama H, Fukuda S, Yoder MC, Pelus LM, Kim KS, Broxmeyer HE (2007) Checkpoint-apoptosis uncoupling in human and mouse embryonic stem cells: a source of karyotpic instability. Blood 109:4518–4527PubMedCrossRefGoogle Scholar
  39. 39.
    Becker KA, Ghule PN, Therrien JA, Lian JB, Stein JL, van Wijnen AJ, Stein GS (2006) Self-renewal of human embryonic stem cells is supported by a shortened G1 cell cycle phase. J Cell Physiol 209:883–893PubMedCrossRefGoogle Scholar
  40. 40.
    Vallier L, Alexander M, Pedersen RA (2005) Activin/Nodal and FGF pathways cooperate to maintain pluripotency of human embryonic stem cells. J Cell Sci 118:4495–4509PubMedCrossRefGoogle Scholar
  41. 41.
    Li J, Wang G, Wang C, Zhao Y, Zhang H, Tan Z, Song Z, Ding M, Deng H (2007) MEK/ERK signaling contributes to the maintenance of human embryonic stem cell self-renewal. Differentiation 75(4):299–307PubMedCrossRefGoogle Scholar
  42. 42.
    Wang L, Schulz TC, Sherrer ES, Dauphin DS, Shin S, Nelson AM, Ware CB, Zhan M, Song CZ, Chen X, Brimble SN, McLean A, Galeano MJ, Uhl EW, D’Amour KA, Chesnut JD, Rao MS, Blau CA, Robins AJ (2007) Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood 110(12):4111–4119PubMedCrossRefGoogle Scholar
  43. 43.
    Xiao L, Yuan X, Sharkis SJ (2006) Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells. Stem Cells 24:1476–1486PubMedCrossRefGoogle Scholar
  44. 44.
    Armstrong L, Hughes O, Yung S, Hyslop L, Stewart R, Wappler I, Peters H, Walter T, Stojkovic P, Evans J, Stojkovic M, Lako M (2006) The role of PI3K/AKT, MAPK/ERK and NFkappabeta signalling in the maintenance of human embryonic stem cell pluripotency and viability highlighted by transcriptional profiling and functional analysis. Hum Mol Genet 15:1894–1913PubMedCrossRefGoogle Scholar
  45. 45.
    Dravid G, Ye Z, Hammond H, Chen G, Pyle A, Donovan P, Yu X, Cheng L (2005) Defining the role of Wnt/beta-catenin signaling in the survival, proliferation, and self-renewal of human embryonic stem cells. Stem Cells 23:1489–1501PubMedCrossRefGoogle Scholar
  46. 46.
    Liu N, Lu M, Tian X, Han Z (2007) Molecular mechanisms involved in self-renewal and pluripotency of embryonic stem cells. J Cell Physiol 211:279–286PubMedCrossRefGoogle Scholar
  47. 47.
    Boiani M, Scholer HR (2005) Regulatory networks in embryo-derived pluripotent stem cells. Nat Rev Mol Cell Biol 6:872–884PubMedCrossRefGoogle Scholar
  48. 48.
    Rao M (2004) Conserved and divergent paths that regulate self-renewal in mouse and human embryonic stem cells. Dev Biol 275:269–286PubMedCrossRefGoogle Scholar
  49. 49.
    Ginis I, Luo Y, Miura T, Thies S, Brandenberger R, Gerecht-Nir S, Amit M, Hoke A, Carpenter MK, Itskovitz-Eldor J, Rao MS (2004) Differences between human and mouse embryonic stem cells. Dev Biol 269:360–380PubMedCrossRefGoogle Scholar
  50. 50.
    Stewart CL, Kaspar P, Brunet LJ, Bhatt H, Gadi I, Kontgen F, Abbondanzo SJ (1992) Blastocyst implantation depends on maternal expression of leukaemia inhibitory factor. Nature 359:76–79PubMedCrossRefGoogle Scholar
  51. 51.
    Ying QL, Nichols J, Chambers I, Smith A (2003) BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115:281–292PubMedCrossRefGoogle Scholar
  52. 52.
    Daheron L, Opitz SL, Zaehres H, Lensch WM, Andrews PW, Itskovitz-Eldor J, Daley GQ (2004) LIF/STAT3 signaling fails to maintain self-renewal of human embryonic stem cells. Stem Cells 22:770–778PubMedCrossRefGoogle Scholar
  53. 53.
    Xu RH, Chen X, Li DS, Li R, Addicks GC, Glennon C, Zwaka TP, Thomson JA (2002) BMP4 initiates human embryonic stem cell differentiation to trophoblast. Nat Biotechnol 20:1261–1264PubMedCrossRefGoogle Scholar
  54. 54.
    Gerami-Naini B, Dovzhenko OV, Durning M, Wegner FH, Thomson JA, Golos TG (2004) Trophoblast differentiation in embryoid bodies derived from human embryonic stem cells. Endocrinology 145:1517–1524PubMedCrossRefGoogle Scholar
  55. 55.
    Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA (2005) Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2:185–190PubMedCrossRefGoogle Scholar
  56. 56.
    Skottman H, Hovatta O (2006) Culture conditions for human embryonic stem cells. Reproduction 132:691–698PubMedCrossRefGoogle Scholar
  57. 57.
    Amit M, Itskovitz-Eldor J (2006) Sources, derivation, and culture of human embryonic stem cells. Semin Reprod Med 24:298–303PubMedCrossRefGoogle Scholar
  58. 58.
    Rajala K, Hakala H, Panula S, Aivio S, Pihlajamaki H, Suuronen R, Hovatta O, Skottman H (2007) Testing of nine different xeno-free culture media for human embryonic stem cell cultures. Hum Reprod 22(5):1231–1238PubMedCrossRefGoogle Scholar
  59. 59.
    Kunath T, Saba-El-Leil MK, Almousailleakh M, Wray J, Meloche S, Smith A (2007) FGF stimulation of the Erk1/2 signalling cascade triggers transition of pluripotent embryonic stem cells from self-renewal to lineage commitment. Development 134:2895–2902PubMedCrossRefGoogle Scholar
  60. 60.
    Herszfeld D, Wolvetang E, Langton-Bunker E, Chung TL, Filipczyk AA, Houssami S, Jamshidi P, Koh K, Laslett AL, Michalska A, Nguyen L, Reubinoff BE, Tellis I, Auerbach JM, Ording CJ, Looijenga LH, Pera MF (2006) CD30 is a survival factor and a biomarker for transformed human pluripotent stem cells. Nat Biotechnol 24:351–357PubMedCrossRefGoogle Scholar
  61. 61.
    Rho JY, Yu K, Han JS, Chae JI, Koo DB, Yoon HS, Moon SY, Lee KK, Han YM (2006) Transcriptional profiling of the developmentally important signalling pathways in human embryonic stem cells. Hum Reprod 21:405–412PubMedCrossRefGoogle Scholar
  62. 62.
    Sperger JM, Chen X, Draper JS, Antosiewicz JE, Chon CH, Jones SB, Brooks JD, Andrews PW, Brown PO, Thomson JA (2003) Gene expression patterns in human embryonic stem cells and human pluripotent germ cell tumors. Proc Natl Acad Sci USA 100:13350–13355PubMedCrossRefGoogle Scholar
  63. 63.
    Brandenberger R, Wei H, Zhang S, Lei S, Murage J, Fisk GJ, Li Y, Xu C, Fang R, Guegler K, Rao MS, Mandalam R, Lebkowski J, Stanton LW (2004) Transcriptome characterization elucidates signaling networks that control human ES cell growth and differentiation. Nat Biotechnol 22:707–716PubMedCrossRefGoogle Scholar
  64. 64.
    James D, Levine AJ, Besser D, Hemmati-Brivanlou A (2005) TGFbeta/activin/nodal signaling is necessary for the maintenance of pluripotency in human embryonic stem cells. Development 132:1273–1282PubMedCrossRefGoogle Scholar
  65. 65.
    Vallier L, Pedersen RA (2005) Human embryonic stem cells: an in vitro model to study mechanisms controlling pluripotency in early mammalian development. Stem Cell Rev 1:119–130PubMedCrossRefGoogle Scholar
  66. 66.
    Peerani R, Rao BM, Bauwens C, Yin T, Wood GA, Nagy A, Kumacheva E, Zandstra PW (2007) Niche-mediated control of human embryonic stem cell self-renewal and differentiation. Embo J 26(22):4744–4755PubMedCrossRefGoogle Scholar
  67. 67.
    Miyazawa K, Shinozaki M, Hara T, Furuya T, Miyazono K (2002) Two major Smad pathways in TGF-beta superfamily signalling. Genes Cells 7:1191–1204PubMedCrossRefGoogle Scholar
  68. 68.
    Besser D (2004) Expression of nodal, lefty-a, and lefty-B in undifferentiated human embryonic stem cells requires activation of Smad2/3. J Biol Chem 279:45076–45084PubMedCrossRefGoogle Scholar
  69. 69.
    Vallier L, Reynolds D, Pedersen RA (2004) Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway. Dev Biol 275:403–421PubMedCrossRefGoogle Scholar
  70. 70.
    Beattie GM, Lopez AD, Bucay N, Hinton A, Firpo MT, King CC, Hayek A (2005) Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23:489–495PubMedCrossRefGoogle Scholar
  71. 71.
    Amit M, Shariki C, Margulets V, Itskovitz-Eldor J (2004) Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod 70:837–845PubMedCrossRefGoogle Scholar
  72. 72.
    Wang L, Li L, Menendez P, Cerdan C, Bhatia M (2005) Human embryonic stem cells maintained in the absence of mouse embryonic fibroblasts or conditioned media are capable of hematopoietic development. Blood 105:4598–4603PubMedCrossRefGoogle Scholar
  73. 73.
    Xu C, Rosler E, Jiang J, Lebkowski JS, Gold JD, O’Sullivan C, Delavan-Boorsma K, Mok M, Bronstein A, Carpenter MK (2005) Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells 23:315–323PubMedCrossRefGoogle Scholar
  74. 74.
    Wang G, Zhang H, Zhao Y, Li J, Cai J, Wang P, Meng S, Feng J, Miao C, Ding M, Li D, Deng H (2005) Noggin and bFGF cooperate to maintain the pluripotency of human embryonic stem cells in the absence of feeder layers. Biochem Biophys Res Commun 330:934–942PubMedCrossRefGoogle Scholar
  75. 75.
    Dvorak P, Dvorakova D, Koskova S, Vodinska M, Najvirtova M, Krekac D, Hampl A (2005) Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells. Stem Cells 23:1200–1211PubMedCrossRefGoogle Scholar
  76. 76.
    Kang HB, Kim JS, Kwon HJ, Nam KH, Youn HS, Sok DE, Lee Y (2005) Basic fibroblast growth factor activates ERK and induces c-fos in human embryonic stem cell line MizhES1. Stem Cells Dev 14:395–401PubMedCrossRefGoogle Scholar
  77. 77.
    Xie T, Spradling AC (1998) decapentaplegic is essential for the maintenance and division of germline stem cells in the Drosophila ovary. Cell 94:251–260PubMedCrossRefGoogle Scholar
  78. 78.
    Tulina N, Matunis E (2001) Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling. Science 294:2546–2549PubMedCrossRefGoogle Scholar
  79. 79.
    Kiger AA, Jones DL, Schulz C, Rogers MB, Fuller MT (2001) Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue. Science 294:2542–2545PubMedCrossRefGoogle Scholar
  80. 80.
    Mayshar Y, Rom E, Chumakov I, Kronman A, Yayon A, Benvenisty N (2008) FGF4 And its novel splice isoform have opposing effects on the maintenance of human embryonic stem cell self renewal. Stem Cells 26(3):767–774PubMedCrossRefGoogle Scholar
  81. 81.
    Martelli AM, Nyakern M, Tabellini G, Bortul R, Tazzari PL, Evangelisti C, Cocco L (2006) Phosphoinositide 3-kinase/Akt signaling pathway and its therapeutical implications for human acute myeloid leukemia. Leukemia 20:911–928PubMedCrossRefGoogle Scholar
  82. 82.
    Pyle AD, Lock LF, Donovan PJ (2006) Neurotrophins mediate human embryonic stem cell survival. Nat Biotechnol 24(3):344–350PubMedCrossRefGoogle Scholar
  83. 83.
    McLean AB, D’Amour KA, Jones KL, Krishnamoorthy M, Kulik MJ, Reynolds DM, Sheppard AM, Liu H, Xu Y, Baetge EE, Dalton S (2007) Activin a efficiently specifies definitive endoderm from human embryonic stem cells only when phosphatidylinositol 3-kinase signaling is suppressed. Stem Cells 25:29–38PubMedCrossRefGoogle Scholar
  84. 84.
    Sidhu KS, Tuch BE (2006) Derivation of three clones from human embryonic stem cell lines by FACS sorting and their characterization. Stem Cells Dev 15:61–69PubMedCrossRefGoogle Scholar
  85. 85.
    Nicholas CR, Gaur M, Wang S, Pera RA, Leavitt AD (2007) A method for single-cell sorting and expansion of genetically modified human embryonic stem cells. Stem Cells Dev 16:109–117PubMedCrossRefGoogle Scholar
  86. 86.
    Hewitt Z, Forsyth NR, Waterfall M, Wojtacha D, Thomson AJ, McWhir J (2006) Fluorescence-activated single cell sorting of human embryonic stem cells. Cloning Stem Cells 8:225–234PubMedCrossRefGoogle Scholar
  87. 87.
    Venable A, Mitalipova M, Lyons I, Jones K, Shin S, Pierce M, Stice S (2005) Lectin binding profiles of SSEA-4 enriched, pluripotent human embryonic stem cell surfaces. BMC Dev Biol 5:15PubMedCrossRefGoogle Scholar
  88. 88.
    Eiges R, Schuldiner M, Drukker M, Yanuka O, Itskovitz-Eldor J, Benvenisty N (2001) Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr Biol 11:514–518PubMedCrossRefGoogle Scholar
  89. 89.
    Rosler ES, Fisk GJ, Ares X, Irving J, Miura T, Rao MS, Carpenter MK (2004) Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn 229:259–274PubMedCrossRefGoogle Scholar
  90. 90.
    Levenstein ME, Ludwig TE, Xu RH, Llanas RA, VanDenHeuvel-Kramer K, Manning D, Thomson JA (2006) Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells 24:568–574PubMedCrossRefGoogle Scholar
  91. 91.
    Walkley CR, Shea JM, Sims NA, Purton LE, Orkin SH (2007) Rb regulates interactions between hematopoietic stem cells and their bone marrow microenvironment. Cell 129:1081–1095PubMedCrossRefGoogle Scholar
  92. 92.
    Perez OD, Nolan GP (2002) Simultaneous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotechnol 20:155–162PubMedGoogle Scholar
  93. 93.
    Sachs K, Perez O, Pe’er D, Lauffenburger DA, Nolan GP (2005) Causal protein-signaling networks derived from multiparameter single-cell data. Science 308:523–529PubMedCrossRefGoogle Scholar
  94. 94.
    Li Y, Powell S, Brunette E, Lebkowski J, Mandalam R (2005) Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products. Biotechnol Bioeng 91:688–698PubMedCrossRefGoogle Scholar
  95. 95.
    Cheon SH, Kim SJ, Jo JY, Ryu WJ, Rhee K, Roh SI (2005) Defined feeder-free culture system of human embryonic stem cells. Biol Reprod 74(3):611PubMedGoogle Scholar
  96. 96.
    Yao S, Chen S, Clark J, Hao E, Beattie GM, Hayek A, Ding S (2006) Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions. Proc Natl Acad Sci USA 103:6907–6912PubMedCrossRefGoogle Scholar
  97. 97.
    Bigdeli N, Andersson M, Strehl R, Emanuelsson K, Kilmare E, Hyllner J, Lindahl A (2007) Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces. J Biotechnol 133(1):146–153PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Morag H. Stewart
    • 1
  • Sean C. Bendall
    • 1
    • 2
  • Mickie Bhatia
    • 1
  1. 1.McMaster Stem Cell and Cancer Research Institute, Michael G. DeGroote School of Medicine, and Department of BiochemistryMcMaster UniversityHamiltonCanada
  2. 2.Don Rix Protein Identification Facility, Department of Biochemistry, Schulich School of Medicine and DentistryUniversity of Western OntarioLondonCanada

Personalised recommendations