Developing defined culture systems for human pluripotent stem cells

Regenerative Medicine

Volumn 6, Issue 5, 2011, Pages 623-634

  Valamehr, Bahram ^a,c   Tsutsui, Hideaki ^b   Ho, Chih Ming ^b   Wu, Hong ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b UNIVERSITY OF CALIFORNIA   (United States)

^c Fate Therapeutics Inc   (United States)

Author keywords

defined culture; feedback system control; feeder free and single cell culture; human embryonic stem cells; human induced pluripotent stem cells; regenerative medicine; small molecules; suspension culture; synthetic surfaces

Indexed keywords

ARGINYLGLYCYLASPARTIC ACID; BONE SIALOPROTEIN; FIBROBLAST GROWTH FACTOR 4; FIBRONECTIN; GANTREZ COPOLYMER; GLYCOGEN SYNTHASE KINASE 3; KRUPPEL LIKE FACTOR 2; KRUPPEL LIKE FACTOR 4; LAMININ; MITOGEN ACTIVATED PROTEIN KINASE; OCTAMER TRANSCRIPTION FACTOR 4; POLYMER; VITRONECTIN;

CELL CULTURE; CELL DIFFERENTIATION; CELL PROLIFERATION; CELL RENEWAL; CELL TYPE; CULTURE MEDIUM; DERIVATIZATION; EMBRYONIC STEM CELL; FETAL CALF SERUM; HUMAN; IN VITRO STUDY; MEDICAL RESEARCH; NONHUMAN; PLURIPOTENT STEM CELL; PRIORITY JOURNAL; PROTEIN EXPRESSION; REVIEW;

CELL CULTURE TECHNIQUES; CELL DIFFERENTIATION; EMBRYONIC STEM CELLS; HUMANS; INDUCED PLURIPOTENT STEM CELLS; REGENERATIVE MEDICINE;

EID: 80052887366     PISSN: 17460751     EISSN: 1746076X     Source Type: Journal    
DOI: 10.2217/rme.11.54     Document Type: Review

References (113)

1. Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154-156 (1981). (Pubitemid 11050741)
2. Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc. Natl Acad. Sci. USA 78, 7634-7638 (1981). (Pubitemid 12186197)
3. Suda Y, Suzuki M, Ikawa Y, Aizawa S. Mouse embryonic stem cells exhibit indefinite proliferative potential. J. Cell. Physiol. 133, 197-201 (1987). (Pubitemid 18022466)
4. Keller G. Embryonic stem cell differentiation: emergence of a new era in biology and medicine. Genes Dev. 19, 1129-1155 (2005). (Pubitemid 40696181)
5. Smith AG. Embryo-derived stem cells: of mice and men. Annu. Rev. Cell Dev. Biol. 17, 435-462 (2001). (Pubitemid 33096183)
6. Beddington RS, Robertson EJ. An assessment of the developmental potential of embryonic stem cells in the midgestation mouse embryo. Development 105, 733-737 (1989). (Pubitemid 19107071)
7. Bradley A, Evans M, Kaufman MH, Robertson E. Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309, 255-256 (1984). (Pubitemid 14139934)
8. Eggan K, Akutsu H, Loring J, et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl Acad. Sci. USA 98, 6209-6214 (2001). (Pubitemid 32488218)
9. Kubiak JZ, Tarkowski AK. Electrofusion of mouse blastomeres. Exp. Cell Res. 157, 561-566 (1985). (Pubitemid 15035407)
10. Jaenisch R. Transgenic animals. Science 240, 1468-1474 (1988).
11. Carver BS, Pandolfi PP. Mouse modeling in oncologic preclinical and translational research. Clin. Cancer Res. 12, 5305-5311 (2006). (Pubitemid 44497243)
12. Hofker MH. Introduction: strategies for developing genetically modified mice. Methods Mol. Biol. 693, 1-10 (2011).
13. Niwa H, Burdon T, Chambers I, Smith A. Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 12, 2048-2060 (1998). (Pubitemid 28323878)
14. Ying QL, Nichols J, Chambers I, Smith A. BMP induction of Id proteins suppresses differentiation and sustains embryonic stem cell self-renewal in collaboration with STAT3. Cell 115, 281-292 (2003). (Pubitemid 37487702)
15. Smith AG, Heath JK, Donaldson DD, et al. Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336, 688-690 (1988). (Pubitemid 19007952)
16. Williams RL, Hilton DJ, Pease S, et al. Myeloid leukaemia inhibitory factor maintains the developmental potential of embryonic stem cells. Nature 336, 684-687 (1988). (Pubitemid 19007951)
17. Smith AG, Hooper ML. Buffalo rat liver cells produce a diffusible activity which inhibits the differentiation of murine embryonal carcinoma and embryonic stem cells. Dev. Biol. 121, 1-9 (1987). (Pubitemid 17076824)
18. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145-1147 (1998). (Pubitemid 28516247)
19. Reubinoff BE, Pera MF, Fong CY, Trounson A, Bongso A. Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399-404 (2000). (Pubitemid 30217526)
20. Burdon T, Chambers I, Stracey C, Niwa H, Smith A. Signaling mechanisms regulating self-renewal and differentiation of pluripotent embryonic stem cells. Cells Tissues Organs 165, 131-143 (1999).
21. Pera MF. Human pluripotent stem cells: a progress report. Curr. Opin. Genet. Dev. 11, 595-599 (2001). (Pubitemid 32800736)
22. Hoffman LM, Carpenter MK. Characterization and culture of human embryonic stem cells. Nat. Biotechnol. 23, 699-708 (2005). (Pubitemid 41716360)
23. Takahashi K, Tanabe K, Ohnuki M, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872 (2007). (Pubitemid 350138099)
24. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676 (2006). (Pubitemid 44233629)
25. Yu J, Vodyanik MA, Smuga-Otto K, et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920 (2007).
26. Hanna J, Wernig M, Markoulaki S, et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318, 1920-1923 (2007).
27. Wernig M, Zhao JP, Pruszak J, et al. Neurons derived from reprogrammed fibroblasts functionally integrate into the fetal brain and improve symptoms of rats with Parkinsons disease. Proc. Natl Acad. Sci. USA 105, 5856-5861 (2008). (Pubitemid 351758462)
28. Yoshida Y, Yamanaka S. iPS cells: a source of cardiac regeneration. J. Mol. Cell. Cardiol. 50, 327-332 (2011).
29. Nelson TJ, Martinez-Fernandez A, Terzic A. Induced pluripotent stem cells: developmental biology to regenerative medicine. Nat. Rev. Cardiol. 7, 700-710 (2010).
30. Plath K, Lowry WE. Progress in understanding reprogramming to the induced pluripotent state. Nat. Rev. Genet. 12, 253-265 (2011).
31. Saha K, Jaenisch R. Technical challenges in using human induced pluripotent stem cells to model disease. Cell Stem Cell 5, 584-595 (2009).
32. Yoshida Y, Yamanaka S. Recent stem cell advances: induced pluripotent stem cells for disease modeling and stem cell-based regeneration. Circulation 122, 80-87 (2010).
33. McDevitt TC, Palecek SP. Innovation in the culture and derivation of pluripotent human stem cells. Curr. Opin. Biotechnol. 19, 527-533 (2008).
34. Skottman H, Narkilahti S, Hovatta O. Challenges and approaches to the culture of pluripotent human embryonic stem cells. Regen. Med. 2(3), 265-273 (2007). (Pubitemid 46845885)
35. Hewitt ZA, Amps KJ, Moore HD. Derivation of GMP raw materials for use in regenerative medicine: hESC-based therapies, progress toward clinical application. Clin. Pharmacol. Ther. 82, 448-452 (2007). (Pubitemid 47414351)
36. Hodges H, Pollock K, Stroemer P, et al. Making stem cell lines suitable for transplantation. Cell Transplant. 16, 101-115 (2007). (Pubitemid 46668786)
37. Lei T, Jacob S, Ajil-Zaraa I, et al. Xeno-free derivation and culture of human embryonic stem cells: current status, problems and challenges. Cell Res. 17, 682-688 (2007). (Pubitemid 47255938)
38. Skottman H, Dilber MS, Hovatta O. The derivation of clinical-grade human embryonic stem cell lines. FEBS Lett. 580, 2875-2878 (2006). (Pubitemid 43729174)
39. Unger C, Skottman H, Blomberg P, Dilber MS, Hovatta O. Good manufacturing practice and clinical-grade human embryonic stem cell lines. Hum. Mol. Genet. 17, R48-R53 (2008).
40. Martin MJ, Muotri A, Gage F, Varki A. Human embryonic stem cells express an immunogenic nonhuman sialic acid. Nat. Med. 11, 228-232 (2005). (Pubitemid 40321360)
41. Ilic D. Culture of human embryonic stem cells and the extracellular matrix microenvironment. Regen. Med. 1, 95-101 (2006).
42. Amit M, Carpenter MK, Inokuma MS, et al. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 227, 271-278 (2000).
43. Eiges R, Schuldiner M, Drukker M, et al. Establishment of human embryonic stem cell-transfected clones carrying a marker for undifferentiated cells. Curr. Biol. 11, 514-518 (2001). (Pubitemid 32305031)
44. Tenzen T, Zembowicz F, Cowan CA. Genome modification in human embryonic stem cells. J. Cell. Physiol. 222, 278-281 (2010).
45. Sathananthan AH, Trounson A. Human embryonic stem cells and their spontaneous differentiation. Ital. J. Anat. Embryol. 110, 151-157 (2005). (Pubitemid 40993970)
46. Pera MF, Trounson AO. Human embryonic stem cells: prospects for development. Development 131, 5515-5525 (2004). (Pubitemid 39625196)
47. Yao S, Chen S, Clark J, et al. Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions. Proc. Natl Acad. Sci. USA 103, 6907-6912 (2006).
48. Furue MK, Na J, Jackson JP, et al. Heparin promotes the growth of human embryonic stem cells in a defined serum-free medium. Proc. Natl Acad. Sci. USA 105, 13409-13414 (2008).
49. Montes R, Ligero G, Sanchez L, et al. Feeder-free maintenance of hESCs in mesenchymal stem cell-conditioned media: distinct requirements for TGF-b and IGF-II. Cell Res. 19, 698-709 (2009).
50. Lu J, Hou R, Booth CJ, Yang SH, Snyder M. Defined culture conditions of human embryonic stem cells. Proc. Natl Acad. Sci. USA 103, 5688-5693 (2006).
51. Ludwig TE, Levenstein ME, Jones JM, et al. Derivation of human embryonic stem cells in defined conditions. Nat. Biotechnol. 24, 185-187 (2006). (Pubitemid 43221701)
52. Pyle AD, Lock LF, Donovan PJ. Neurotrophins mediate human embryonic stem cell survival. Nat. Biotechnol. 24, 344-350 (2006). (Pubitemid 43372159)
53. Ellerstrom C, Strehl R, Moya K, et al. Derivation of a xeno-free human embryonic stem cell line. Stem Cells 24, 2170-2176 (2006). (Pubitemid 44527469)
54. Hovatta O, Mikkola M, Gertow K, et al. A culture system using human foreskin fibroblasts as feeder cells allows production of human embryonic stem cells. Hum. Reprod. 18, 1404-1409 (2003). (Pubitemid 36840945)
55. Richards M, Fong CY, Chan WK, Wong PC, Bongso A. Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat. Biotechnol. 20, 933-936 (2002). (Pubitemid 35001132)
56. Richards M, Tan S, Fong CY, et al. Comparative evaluation of various human feeders for prolonged undifferentiated growth of human embryonic stem cells. Stem Cells 21, 546-556 (2003). (Pubitemid 37444111)
57. Stojkovic P, Lako M, Stewart R, et al. An autogeneic feeder cell system that efficiently supports growth of undifferentiated human embryonic stem cells. Stem Cells 23, 306-314 (2005). (Pubitemid 40404449)
58. Wang Q, Fang ZF, Jin F, et al. Derivation and growing human embryonic stem cells on feeders derived from themselves. Stem Cells 23, 1221-1227 (2005). (Pubitemid 41504909)
59. Stacey GN, Cobo F, Nieto A, et al. The development of feeder cells for the preparation of clinical grade hES cell lines: challenges and solutions. J. Biotechnol. 125, 583-588 (2006). (Pubitemid 44333118)
60. Xu C, Inokuma MS, Denham J, et al. Feeder-free growth of undifferentiated human embryonic stem cells. Nat. Biotechnol. 19, 971-974 (2001). (Pubitemid 32947578)
61. Chin AC, Fong WJ, Goh LT, et al. Identification of proteins from feeder conditioned medium that support human embryonic stem cells. J. Biotechnol. 130, 320-328 (2007). (Pubitemid 46920536)
62. Cormier JT, zur Nieden NI, Rancourt DE, Kallos MS. Expansion of undifferentiated murine embryonic stem cells as aggregates in suspension culture bioreactors. Tissue Eng. 12, 3233-3245 (2006). (Pubitemid 44846427)
63. Amit M, Laevsky I, Miropolsky Y, et al. Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells. Nat. Protoc. 6, 572-579 (2011).
64. Zweigerdt R, Olmer R, Singh H, Haverich A, Martin U. Scalable expansion of human pluripotent stem cells in suspension culture. Nat. Protoc. 6, 689-700 (2011).
65. Olmer R, Haase A, Merkert S, et al. Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium. Stem Cell Res. 5, 51-64 (2010).
66. Singh H, Mok P, Balakrishnan T, Rahmat SNB, Zweigerdt R. Up-scaling single cell-inoculated suspension culture of human embryonic stem cells. Stem Cell Res. 4, 165-179 (2010).
67. Steiner D, Khaner H, Cohen M, et al. Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat. Biotechnol. 28, 361-364 (2010).
68. Amit M, Shariki C, Margulets V, Itskovitz-Eldor J. Feeder layer-and serum-free culture of human embryonic stem cells. Biol. Reprod. 70, 837-845 (2004). (Pubitemid 38233724)
69. Beattie GM, Lopez AD, Bucay N, et al. Activin A maintains pluripotency of human embryonic stem cells in the absence of feeder layers. Stem Cells 23, 489-495 (2005). (Pubitemid 40478488)
70. Li Y, Powell S, Brunette E, Lebkowski J, Mandalam R. Expansion of human embryonic stem cells in defined serum-free medium devoid of animal-derived products. Biotechnol. Bioeng. 91, 688-698 (2005). (Pubitemid 41283861)
71. Liu Y, Song Z, Zhao Y, 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, 131-139 (2006). (Pubitemid 43865977)
72. Xiao L, Yuan X, Sharkis SJ. Activin A maintains self-renewal and regulates fibroblast growth factor, Wnt, and bone morphogenic protein pathways in human embryonic stem cells. Stem Cells 24, 1476-1486 (2006). (Pubitemid 44464664)
73. Hasegawa K, Fujioka T, Nakamura Y, Nakatsuji N, Suemori H. A method for the selection of human embryonic stem cell sublines with high replating efficiency after single-cell dissociation. Stem Cells 24, 2649-2660 (2006). (Pubitemid 46053003)
74. Watanabe K, Ueno M, Kamiya D, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681-686 (2007). (Pubitemid 46897207)
75. Chen G, Hou Z, Gulbranson DR, Thomson JA. Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell 7, 240-248 (2010).
76. Damoiseaux R, Sherman SP, Alva JA, Peterson C, Pyle AD. Integrated chemical genomics reveals modifiers of survival in human embryonic stem cells. Stem Cells 27, 533-542 (2009).
77. Ohgushi M, Matsumura M, Eiraku M, et al. Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7, 225-239 (2010).
78. Ohgushi M, Sasai Y. Lonely death dance of human pluripotent stem cells: ROCKing between metastable cell states. Trends Cell Biol. 21(5), 274-282 (2011).
79. Walker A, Su H, Conti MA, et al. Non-muscle myosin II regulates survival threshold of pluripotent stem cells. Nat. Commun. 1, 71 (2010).
80. Xu Y, Zhu X, Hahm HS, et al. Revealing a core signaling regulatory mechanism for pluripotent stem cell survival and self-renewal by small molecules. Proc. Natl Acad. Sci. USA 107, 8129-8134 (2010).
81. Ardehali R, Inlay MA, Ali SR, et al. Overexpression of BCL2 enhances survival of human embryonic stem cells during stress and obviates the requirement for serum factors. Proc. Natl Acad. Sci. USA 108, 3282-3287 (2011).
82. Nakayama M. Homologous recombination in human iPS and ES cells for use in gene correction therapy. Drug Discov. Today 15, 198-202 (2010).
83. Stewart MH, Bendall SC, Bhatia M. Deconstructing human embryonic stem cell cultures: niche regulation of self-renewal and pluripotency. J. Mol. Med. 86, 875-886 (2008).
84. Ying QL, Wray J, Nichols J, et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519-523 (2008). (Pubitemid 351733324)
85. Silva J, Smith A. Capturing pluripotency. Cell 132, 532-536 (2008). (Pubitemid 351253697)
86. Buehr M, Meek S, Blair K, et al. Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287-1298 (2008).
87. Li P, Tong C, Mehrian-Shai R, et al. Germline competent embryonic stem cells derived from rat blastocysts. Cell 135, 1299-1310 (2008).
88. Kawamata M, Ochiya T. Generation of genetically modified rats from embryonic stem cells. Proc. Natl Acad. Sci. USA 107, 14223-14228 (2010).
89. Tsutsui H, Valamehr B, Hindoyan A, et al. An optimized small molecule inhibitor cocktail supports long-human embryonic stem cells. Nat. Commun. 2, 167 (2011).
90. Hanna J, Cheng AW, Saha K, et al. Human embryonic stem cells with biological and epigenetic characteristics similar to those of mouse ESCs. Proc. Natl Acad. Sci. USA 107, 9222-9227 (2010).
91. Sun CP, Usui T, Yu F, et al. Integrative systems control approach for reactivating Kaposis sarcoma-associated herpesvirus (KSHV) with combinatory drugs. Integr. Biol. 1, 123-130 (2009).
92. Wong PK, Yu F, Shahangian A, et al. Closed-loop control of cellular functions using combinatory drugs guided by a stochastic search algorithm. Proc. Natl Acad. Sci. USA 105, 5105-5110 (2008). (Pubitemid 351738517)
93. Braam SR, Zeinstra L, Litjens S, et al. Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via aVb5 integrin. Stem Cells 26, 2257-2265 (2008).
94. Miyazaki T, Futaki S, Hasegawa K, et al. Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem. Biophys. Res. Commun. 375, 27-32 (2008).
95. Rodin S, Domogatskaya A, Strom S, et al. Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nat. Biotechnol. 28, 611-615 (2010).
96. Rowland TJ, Miller LM, Blaschke AJ, et al. Roles of integrins in human induced pluripotent stem cell growth on Matrigel and vitronectin. Stem Cells Dev. 19, 1231-1240 (2010).
97. Nagaoka M, Si-Tayeb K, Akaike T, Duncan SA. Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum. BMC Dev. Biol. 10, 60 (2010).
98. Melkoumian Z, Weber JL, Weber DM, et al. Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells. Nat. Biotechnol. 28, 606-610 (2010).
99. Klim JR, Li LY, Wrighton PJ, Piekarczyk MS, Kiessling LL. A defined glycosaminoglycan-binding substratum for human pluripotent stem cells. Nat. Methods 7, 989-994 (2010).
100. Villa-Diaz LG, Nandivada H, Ding J, et al. Synthetic polymer coatings for long-term growth of human embryonic stem cells. Nat. Biotechnol. 28, 581-583 (2010).
101. Brafman DA, Chang CW, Fernandez A, et al. Long-term human pluripotent stem cell self-renewal on synthetic polymer surfaces. Biomaterials 31, 9135-9144 (2010).
102. Mei Y, Saha K, Bogatyrev SR, et al. Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells. Nat. Mater. 9, 768-778 (2010).
103. Chowdhury F, Li YZ, Poh YC, et al. Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions. PLoS One 5, e15655 (2010).
104. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 126, 677-689 (2006). (Pubitemid 44233625)
105. Taapken SM, Nisler BS, Newton MA, et al. Karotypic abnormalities in human induced pluripotent stem cells and embryonic stem cells. Nat. Biotechnol. 29, 313-314 (2011).
106. Mitalipova MM, Rao RR, Hoyer DM, et al. Preserving the genetic integrity of human embryonic stem cells. Nat. Biotechnol. 23, 19-20 (2005). (Pubitemid 41724618)
107. Akopian V, Andrews PW, Beil S, et al. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells. In Vitro Cell. Dev. Biol. Anim. 46, 247-258 (2010).
108. Chen G, Gulbranson DR, Hou Z, et al. Chemically defined conditions for human iPSC derivation and culture. Nat. Methods 8, 424-429 (2011).
109. Lock LT, Tzanakakis ES. Expansion and differentiation of human embryonic stem cells to endoderm progeny in a microcarrier stirred-suspension culture. Tissue Eng. Part A 15, 2051-2063 (2009).
110. Nie Y, Bergendahl V, Hei DJ, Jones JM, Palecek SP. Scalable culture and cryopreservation of human embryonic stem cells on microcarriers. Biotechnol. Prog. 25, 20-31 (2009).
111. Oh SKW, Chen AK, Mok Y, et al. Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Res. 2, 219-230 (2009).
112. Phillips BW, Horne R, Lay TS, et al. Attachment and growth of human embryonic stem cells on microcarriers. J. Biotechnol. 138, 24-32 (2008).
113. Serra M, Brito C, Sousa MFQ, et al. Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control. J. Biotechnol. 148, 208-215 (2010).