人類多能性幹細胞擁有特殊的潛在治療應用，可以助於損壞的器官修復及疾病的治療。然而，臨床實驗的測試受限於細胞培養於老鼠纖維母細胞培養層。在未來，必須開發一個能培養人類多能性幹細胞之無外源汙染的材料。無滋養層培養是利用合成生醫材料接枝奈米片段作為幹細胞的培養盤，他可以幫助人類多能性幹細胞 (人類胚胎幹細胞及人類誘導型多能性幹細胞) 繁殖且同時在無滋養層及外源汙染的培養下維持其多能性。
本人研究在無老鼠纖維母細胞之滋養層下培養人類多能性幹細胞在聚乙烯醇-共-衣康酸 (PVA-IA) 表面接枝奈米片段 (KGGPQVTRGDVFTMP [cell-binding domain derived from vitronectin, oligoVN]) 能夠生長且維持其多能性。利用PVA-IA薄膜表面接枝高濃度之寡肽纖粘蛋白且具有最適合軟硬度的培養盤培養人類多能性幹細胞，比較於市面世上販售合成生醫材料 (Synthemax II)，細胞表現較高的貼覆率及成長倍率且較低的分化比率。
更者，經10代培養人類胚胎幹細胞 (WA09) 及人類誘導型多能性幹細胞 (HPS0077) 於PVA-IA塗層接枝1000µg/mL的寡肽纖粘蛋白且具有理想軟硬度25.3kPa的表面，其擁有極好的多能性蛋白及分化蛋白表現。此結果指出人類多能性幹細胞可以培養於PVA-IA培養系統且維持多能性。在未來，PVA-IA培養系統可以被用於在無外源汙染及無滋養層的環境下利用人類原始的組織來製造人類誘導型多能性幹細胞。

Human pluripotent stem cells (hPSCs) have significant potential in therapeutic applications for damaged organs or diseases. However, the tentative clinical potential of hPSCs is restricted by the use of mouse embryonic fibroblasts (MEFs) as a feeder layer. It is necessary to develop a suitable culture system without using xeno-contaminated materials for hPSCs culture in future. The feeder-free cultures using synthetic biomaterials having nanosegments as stem cell culture materials can support the propagation of human pluripotent stem cells (hPSCs), human embryonic stem cells (hESCs), and induced pluripotent stem cells (hiPSCs) while maintaining pluripotency in feeder-free and xeno-free cultures.
I investigated that hPSCs could proliferate and keep pluripotenty without usage of a feeder layer of MEFs where hPSCs were cultured on polyvinylalcohol-co-itaconic acid (PVA-IA) grafted with nanosegment (KGGPQVTRGDVFTMP [cell-binding domain derived from vitronectin, oligoVN]). The hPSCs on PVA-IA film grafted with high concentration of oligoVN having optimal elasticity showed higher colony attachment ratio, higher colony expansion fold, and lower differentiation ratio compared to those on commercially availiable synthetic biomaterial (Synethemax II).
Moreover, hESCs (WA09) and hiPSCs (HPS0077) cultured on PVA-IA films grafted with 1000µg/ml of oligo-VN with 25.3kPa elasticity after 10 passage showed excellent pluripotent protein and differentiation protein expression in embryoid boid. This result indicates that hPSCs can be cultured and maintain their pluripotency on PVA-IA culture system. In future, the PVA-IA culture system could be used to generate hiPSCs from primary human tissue cells on xeno-free and feeder-free conditions.

1. Andrews, P.W., et al., Embryonic stem (ES) cells and embryonal carcinoma (EC) cells: opposite sides of the same coin. Biochem Soc Trans, 2005. 33(Pt 6): p. 1526-30.
2. Evans, M.J. and M.H. Kaufman, Establishment in culture of pluripotential cells from mouse embryos. Nature, 1981. 292(5819): p. 154-6.
3. Embryonic and Induced Pluripotent Stem Cells, Sigma-Aldrich, http://www.sigmaaldrich.com/life-science/stem-cell-biology/ipsc.html,6/6/2011 15:05.
4. Thomson, J.A., et al., Embryonic stem cell lines derived from human blastocysts. Science, 1998. 282(5391): p. 1145-7.
5. Mitalipov, S. and D. Wolf, Totipotency, pluripotency and nuclear reprogramming. Adv Biochem Eng Biotechnol, 2009. 114: p. 185-99.
6. Campbell, K.H., et al., Sheep cloned by nuclear transfer from a cultured cell line. Nature, 1996. 380(6569): p. 64-6.
7. Palmarini, M., A veterinary twist on pathogen biology. PLoS Pathog, 2007. 3(2): p. e12.
8. Shiels, P.G., et al., Analysis of telomere lengths in cloned sheep. Nature, 1999. 399(6734): p. 316-7
9. Tachibana, M., et al., Human embryonic stem cells derived by somatic cell nuclear transfer. Cell, 2013. 153(6): p. 1228-38.
10. Chung, Y.G., et al., Human Somatic Cell Nuclear Transfer Using Adult Cells. Cell Stem Cell, 2014.7. Mitalipov, S. and D. Wolf, Totipotency, pluripotency and nuclear reprogramming. Adv Biochem Eng Biotechnol, 2009. 114: p. 185-99.
11. Engler, A.J., et al., Matrix elasticity directs stem cell lineage specification. Cell, 2006. 126(4): p. 677-89.
12. Shih, Y. R. V.; Tseng, K. F.; Lai, H. Y.; Lin, C. H.; Lee, O. K. J.Bone Miner. Res. 2011, 26, 730.
13. Engler, A.J., et al., Matrix elasticity directs stem cell lineage specification. Cell, 2006. 126(4): p. 677-89.
14. Aguilar, H.N. and B.F. Mitchell, Physiological pathways and molecular mechanisms regulating uterine contractility. Hum Reprod Update, 2010. 16(6): p. 725-44
15. Kovács, M., et al., Mechanism of blebbistatin inhibition of myosin II. Journal of Biological Chemistry, 2004. 279(34): p. 35557-35563.
16. Ilic D, Stephenson E, Wood V, Jacquet L, Stevenson D, Petrova A,Kadeva N, Codognotto S, Patel H, Semple M, Cornwell G, OgilvieC, Braude P. Derivation and feeder-free propagation of humanembryonic stem cells under xeno-free conditions. Cytotherapy2012;14:122–8. regulating uterine contractility. Hum Reprod Update, 2010. 16(6): p. 725-44
17. Abraham S, Sheridan SD, Miller B, Rao RR. Stable propagation ofhuman embryonic and induced pluripotent stem cells on decellu-larized human substrates. Biotechnol Prog 2010;26:1126–34.
18. Fu X, Toh WS, Liu H, Lu K, Li M, Cao T. Establishment of clinicallycompliant human embryonic stem cells in an autologous feeder-free system. Tissue Eng C 2011;17:927–37.
19. Meng G, Liu S, Li X, Krawetz R, Rancourt DE. Extracellularmatrix isolated from foreskin fibroblasts supports long-termxeno-free human embryonic stem cell culture. Stem Cells Dev2010;19:547–56.
20. Stelling MP, Lages YM, Tovar AM, Mourao PA, Rehen SK. Matrix-bound heparan sulfate is essential for the growth and pluripotencyof human embryonic stem cells. Glycobiology 2013;23:337–45.
21. Higuchi A, Ling QD, Ko YA, Chang Y, Umezawa A. Biomaterials forthe feeder-free culture of human embryonic stem cells and inducedpluripotent stem cells. Chem Rev 2011;111:3021–35.
22. Rajala K, Hakala H, Panula S, Aivio S, Pihlajamaki H, Suuronen R,Hovatta O, Skottman H. Testing of nine different xeno-free cul-ture media for human embryonic stem cell cultures. Hum Reprod2007;22:1231–8.
23. Manton KJ, Richards S, Van Lonkhuyzen D, Cormack L, Leavesley D,Upton Z. A chimeric vitronectin: IGF-I protein supports feeder-cell-free and serum-free culture of human embryonic stem cells. StemCells Dev 2010;19:1297–305.
24. Zonca Jr MR, Yune PS, Heldt CL, Belfort G, Xie Y. High-throughputscreening of substrate chemistry for embryonic stem cell attach-ment, expansion, and maintaining pluripotency. Macromol Biosci2013;13:177–90.
25. Meng GL, Liu SY, Rancourt DE. Synergistic effect of medium, matrix,and exogenous factors on the adhesion and growth of humanpluripotent stem cells under defined, xeno-free conditions. StemCells Dev 2012;21:2036–48.
26. Higuchi A, Ling QD, Hsu ST, Umezawa A. Biomimetic cell cul-ture proteins as extracellular matrices for stem cell differentiation.Chem Rev 2012;112:4507–40.
27. Gumbiner, B.M., Regulation of cadherin-mediated adhesion in morphogenesis. Nat Rev Mol Cell Biol, 2005. 6(8): p. 622-34.
28. Higuchi, A., et al., Biomaterials for the feeder-free culture of human embryonic stem cells and induced pluripotent stem cells. Chem Rev, 2011. 111(5): p. 3021-35.
29. Ullmann, U., et al., Epithelial-mesenchymal transition process in human embryonic stem cells cultured in feeder-free conditions. Mol Hum Reprod, 2007. 13(1): p. 21-32.
30. Nagaoka, M., et al., Culture of human pluripotent stem cells using completely defined conditions on a recombinant E-cadherin substratum. Bmc Developmental Biology, 2010. 10.
31. Dedhar, S., Cell-substrate interactions and signaling through ILK. Current Opinion in Cell Biology, 2000. 12(2): p. 250-256.
32. Pashuck, E.T. and M.M. Stevens, Designing Regenerative Biomaterial Therapies for the Clinic. Science Translational Medicine, 2012. 4(160).
33. Stephenson, E., et al., Derivation and propagation of human embryonic stem cell lines from frozen embryos in an animal product-free environment. Nature Protocols, 2012. 7(7): p. 1366-1381.
34. Li, Z.S., et al., Feeder-free self-renewal of human embryonic stem cells in 3D porous natural polymer scaffolds. Biomaterials, 2010. 31(3): p. 404-412.
35. Liu, Y.X., et al., Modified Hyaluronan Hydrogels Support the Maintenance of Mouse Embryonic Stem Cells and Human Induced Pluripotent Stem Cells. Macromolecular Bioscience, 2012. 12(8): p. 1034-1042.
36. Li, Z.S., et al., Feeder-free self-renewal of human embryonic stem cells in 3D porous natural polymer scaffolds. Biomaterials, 2010. 31(3): p. 404-412.
37. Siti-Ismail, N., et al., The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance. Biomaterials, 2008. 29(29): p. 3946-3952.
38. Klim, J.R., et al., A defined glycosaminoglycan-binding substratum for human pluripotent stem cells. Nature Methods, 2010. 7(12): p. 989-U72.
39. Gerecht, S., et al., Hyaluronic acid hydrogel for controlled self-renewal and differentiation of human embryonic stem cells. Proc Natl Acad Sci U S A, 2007. 104(27): p. 11298-303.
40. Nandivada, H., et al., Fabrication of synthetic polymer coatings and their use in feeder-free culture of human embryonic stem cells. Nature Protocols, 2011. 6(7): p. 1037-1043.
41. Mei, Y., et al., Combinatorial development of biomaterials for clonal growth of human pluripotent stem cells. Nature Materials, 2010. 9(9): p. 768-778.
42. Mahlstedt, M.M., et al., Maintenance of Pluripotency in Human Embryonic Stem Cells Cultured on a Synthetic Substrate in Conditioned Medium. Biotechnology and Bioengineering, 2010. 105(1): p. 130-140.
43. Nie, Y., et al., Scalable Culture and Cryopreservation of Human Embryonic Stem Cells on Microcarriers. Biotechnology Progress, 2009. 25(1): p. 20-31.
44. Kim, S., et al., A novel culture technique for human embryonic stem cells using porous membranes. Stem Cells, 2007. 25(10): p. 2601-2609.
45. Bigdeli, N., et al., Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces. Journal of Biotechnology, 2008. 133(1): p. 146-153.
46. Villa-Diaz, L.G., et al., Synthetic polymer coatings for long-term growth of human embryonic stem cells. Nature Biotechnology, 2010. 28(6): p. 581-583.
47. Brafman, D.A., et al., Long-term human pluripotent stem cell self-renewal on synthetic polymer surfaces. Biomaterials, 2010. 31(34): p. 9135-9144.
48. Nandivada, H., et al., Fabrication of synthetic polymer coatings and their use in feeder-free culture of human embryonic stem cells. Nature Protocols, 2011. 6(7): p. 1037-1043.
49. Irwin, E.E., et al., Engineered polymer-media interfaces for the long-term self-renewal of human embryonic stem cells. Biomaterials, 2011. 32(29): p. 6912-6919.
50. Ross, A.M., et al., Synthetic substrates for long-term stem cell culture. Polymer, 2012. 53(13): p. 2533-2539.
51. Zhang, R., et al., A thermoresponsive and chemically defined hydrogel for long-term culture of human embryonic stem cells. Nature Communications, 2013. 4.
52. Higuchi, A., et al., Photon-modulated changes of cell attachments on poly(spiropyran-co-methyl methacrylate) membranes. Biomacromolecules, 2004. 5(5): p. 1770-4.
53. Higuchi, A., et al., Temperature-dependent cell detachment on Pluronic gels. Biomacromolecules, 2005. 6(2): p. 691-6.
54. Tamura, A., et al., Temperature-responsive poly(N-isopropylacrylamide)-grafted microcarriers for large-scale non-invasive harvest of anchorage-dependent cells. Biomaterials, 2012. 33(15): p. 3803-12.
55. Saito, T., et al., Reversal of Diabetes by the Creation of Neo-Islet Tissues Into a Subcutaneous Site Using Islet Cell Sheets. Transplantation, 2011. 92(11): p. 1231-1236.
56. Wei, H., et al., Thermo-sensitive polymeric micelles based on poly(N-isopropylacrylamide) as drug carriers. Progress in Polymer Science, 2009. 34(9): p. 893-910.
57. Carlson, A.L., et al., Microfibrous substrate geometry as a critical trigger for organization, self-renewal, and differentiation of human embryonic stem cells within synthetic 3-dimensional microenvironments. Faseb Journal, 2012. 26(8): p. 3240-3251.
58. Kraehenbuehl, T.P., R. Langer, and L.S. Ferreira, Three-dimensional biomaterials for the study of human pluripotent stem cells. Nat Methods, 2011. 8(9): p. 731-6.
59. Meng, G., S. Liu, and D.E. Rancourt, Synergistic effect of medium, matrix, and exogenous factors on the adhesion and growth of human pluripotent stem cells under defined, xeno-free conditions. Stem Cells Dev, 2012. 21(11): p. 2036-48.
60. Braam, S.R., et al., Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alpha V beta 5 integrin. Stem Cells, 2008. 26(9): p. 2257-2265.
61. Rodin, S., et al., Long-term self-renewal of human pluripotent stem cells on human recombinant laminin-511. Nature Biotechnology, 2010. 28(6): p. 611-U102.
62. Li, J.A., et al., Impact of vitronectin concentration and surface properties on the stable propagation of human embryonic stem cells. Biointerphases, 2010. 5(3): p. Fa132-Fa142.
63. Nishishita, N., et al., Generation of Virus-Free Induced Pluripotent Stem Cell Clones on a Synthetic Matrix via a Single Cell Subcloning in the Naive State. Plos One, 2012. 7(6).
64. Kim, B.S., et al., Design of artificial extracellular matrices for tissue engineering. Progress in Polymer Science, 2011. 36(2): p. 238-268.
65. Melkoumian, Z., et al., Synthetic peptide-acrylate surfaces for long-term self-renewal and cardiomyocyte differentiation of human embryonic stem cells. Nature Biotechnology, 2010. 28(6): p. 606-U95.
66. Hoffman, L.M. and M.K. Carpenter, Characterization and culture of human embryonic stem cells. Nature Biotechnology, 2005. 23(6): p. 699-708.
67. Kolhar, P., et al., Synthetic surfaces for human embryonic stem cell culture. Journal of Biotechnology, 2010. 146(3): p. 143-146.
68. Klim, J.R., et al., A defined glycosaminoglycan-binding substratum for human pluripotent stem cells. Nature Methods, 2010. 7(12): p. 989-U72.
69. Meng, G., S. Liu, and D.E. Rancourt, Synergistic effect of medium, matrix, and exogenous factors on the adhesion and growth of human pluripotent stem cells under defined, xeno-free conditions. Stem Cells Dev, 2012. 21(11): p. 2036-48.
70. Phillips, B., et al., Attachment and growth of human embryonic stem cells on microcarriers (vol 138, pg 24, 2008). Journal of Biotechnology, 2009. 139(2): p. 194-194.
71. Janssens, S., et al., Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. Lancet, 2006. 367(9505): p. 113-121.
72. Serra, M., et al., Microencapsulation technology: a powerful tool for integrating expansion and cryopreservation of human embryonic stem cells. PLoS One, 2011. 6(8): p. e23212.
73. Steiner, D., et al., Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension. Nat Biotechnol, 2010. 28(4): p. 361-4.
74. Amit, M., et al., Suspension Culture of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells. Stem Cell Reviews and Reports, 2010. 6(2): p. 248-259.
75. Olmer, R., et al., Long term expansion of undifferentiated human iPS and ES cells in suspension culture using a defined medium. Stem Cell Research, 2010. 5(1): p. 51-64.
76. Zweigerdt, R., et al., Scalable expansion of human pluripotent stem cells in suspension culture. Nature Protocols, 2011. 6(5): p. 689-700.
77. Amit, M., et al., Dynamic suspension culture for scalable expansion of undifferentiated human pluripotent stem cells. Nature Protocols, 2011. 6(5): p. 572-579.
78. Larijani, M.R., et al., Long-Term Maintenance of Undifferentiated Human Embryonic and Induced Pluripotent Stem Cells in Suspension. Stem Cells and Development, 2011. 20(11): p. 1911-1923.
79. Marinho, P.A.N., et al., Xeno-Free Production of Human Embryonic Stem Cells in Stirred Microcarrier Systems Using a Novel Animal/Human-Component-Free Medium. Tissue Engineering Part C-Methods, 2013. 19(2): p. 146-155.
80. Chen, A.K., et al., Critical microcarrier properties affecting the expansion of undifferentiated human embryonic stem cells. Stem Cell Res, 2011. 7(2): p. 97-111.
81. Fernandes, A.M., et al., Successful scale-up of human embryonic stem cell production in a stirred microcarrier culture system. Brazilian Journal of Medical and Biological Research, 2009. 42(6): p. 515-522.
82. Serra, M., et al., Improving expansion of pluripotent human embryonic stem cells in perfused bioreactors through oxygen control. Journal of Biotechnology, 2010. 148(4): p. 208-215.
83. Oh, S.K.W., et al., Long-term microcarrier suspension cultures of human embryonic stem cells. Stem Cell Research, 2009. 2(3): p. 219-230.
84. Bardy, J., et al., Microcarrier Suspension Cultures for High-Density Expansion and Differentiation of Human Pluripotent Stem Cells to Neural Progenitor Cells. Tissue Engineering Part C-Methods, 2013. 19(2): p. 166-180.
85. Storm, M.P., et al., Three-Dimensional Culture Systems for the Expansion of Pluripotent Embryonic Stem Cells. Biotechnology and Bioengineering, 2010. 107(4): p. 683-695.
86. Lock, L.T. and E.S. Tzanakakis, Expansion and Differentiation of Human Embryonic Stem Cells to Endoderm Progeny in a Microcarrier Stirred-Suspension Culture. Tissue Engineering Part A, 2009. 15(8): p. 2051-2063.
87. Heng, B.C., et al., Translating Human Embryonic Stem Cells from 2-Dimensional to 3-Dimensional Cultures in a Defined Medium on Laminin- and Vitronectin-Coated Surfaces. Stem Cells and Development, 2012. 21(10): p. 1701-1715.
88. Nie, Y., et al., Scalable Culture and Cryopreservation of Human Embryonic Stem Cells on Microcarriers. Biotechnology Progress, 2009. 25(1): p. 20-31
89. Tamura, A., et al., Temperature-responsive poly(N-isopropylacrylamide)-grafted microcarriers for large-scale non-invasive harvest of anchorage-dependent cells. Biomaterials, 2012. 33(15): p. 3803-12.
90. Ko, D.Y., et al., Recent progress of in situ formed gels for biomedical applications. Progress in Polymer Science, 2013. 38(3-4): p. 672-701.
91. Lee, K.Y. and D.J. Mooney, Alginate: properties and biomedical applications. Prog Polym Sci, 2012. 37(1): p. 106-126.
92. Huang, X.B., et al., Microenvironment of alginate-based microcapsules for cell culture and tissue engineering. Journal of Bioscience and Bioengineering, 2012. 114(1): p. 1-8.
93. Siti-Ismail, N., et al., The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance. Biomaterials, 2008. 29(29): p. 3946-3952.
94. Wilson, J.L. and T.C. McDevitt, Stem cell microencapsulation for phenotypic control, bioprocessing, and transplantation. Biotechnology and Bioengineering, 2013. 110(3): p. 667-682.
95. Jang, M., et al., A feeder-free, defined three-dimensional polyethylene glycol-based extracellular matrix niche for culture of human embryonic stem cells. Biomaterials, 2013. 34(14): p. 3571-3580.
96. Lutolf, M.R., et al., Repair of bone defects using synthetic mimetics of collagenous extracellular matrices. Nature Biotechnology, 2003. 21(5): p. 513-518.
97. Higuchi, A., et al., Physical cues of biomaterials guide stem cell differentiation fate. Chem Rev, 2013. 113(5): p. 3297-328.
98. Ameen, C., et al., Human embryonic stem cells: current technologies and emerging industrial applications. Crit Rev Oncol Hematol, 2008. 65(1): p. 54-80.
99. Higuchi, A., et al., Design of polymeric materials for culturing human pluripotent stem cells: Progress toward feeder-free and xeno-free culturing. Progress in Polymer Science, 2014.
100. Thomson, J.A., et al., Embryonic stem cell lines derived from human blastocysts. Science, 1998. 282(5391): p. 1145-7.
101. Takahashi, K., et al., Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 2007. 131(5): p. 861-72.
102. Brimble, S.N., et al., Karyotypic stability, genotyping, differentiation, feeder-free maintenance, and gene expression sampling in three human embryonic stem cell lines derived prior to August 9, 2001. Stem Cells Dev, 2004. 13(6): p. 585-97.
103. Xu, C., et al., Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells, 2005. 23(3): p. 315-23.
104. Phillips, B.W., et al., Attachment and growth of human embryonic stem cells on microcarriers. J Biotechnol, 2008. 138(1-2): p. 24-32.
105. Bigdeli, N., et al., Adaptation of human embryonic stem cells to feeder-free and matrix-free culture conditions directly on plastic surfaces. J Biotechnol, 2008. 133(1): p. 146-53.
106. Baxter, M.A., et al., Analysis of the distinct functions of growth factors and tissue culture substrates necessary for the long-term self-renewal of human embryonic stem cell lines. Stem Cell Res, 2009. 3(1): p. 28-38.
107. Amit, M., et al., Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod, 2004. 70(3): p. 837-45.
108. O'Connor, M.D., et al., Alkaline phosphatase-positive colony formation is a sensitive, specific, and quantitative indicator of undifferentiated human embryonic stem cells. Stem Cells, 2008. 26(5): p. 1109-16.
109. Kokubu, F., et al., Diverse organization of immunoglobulin VH gene loci in a primitive vertebrate. EMBO J, 1988. 7(11): p. 3413-22.
110. Pera, M.F., B. Reubinoff, and A. Trounson, Human embryonic stem cells. J Cell Sci, 2000. 113 ( Pt 1): p. 5-10.
111. Andrews, P.W., et al., Two monoclonal antibodies recognizing determinants on human embryonal carcinoma cells react specifically with the liver isozyme of human alkaline phosphatase. Hybridoma, 1984. 3(1): p. 33-9.
112. Amit, M., et al., Feeder layer- and serum-free culture of human embryonic stem cells. Biol Reprod, 2004. 70(3): p. 837-45.
113. Yamanaka, S., et al., Pluripotency of embryonic stem cells. Cell Tissue Res, 2008. 331(1): p. 5-22.
114. Xu, C., et al., Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol, 2001. 19(10): p. 971-4.
115. Harkness, L., et al., Isolation and differentiation of chondrocytic cells derived from human embryonic stem cells using dlk1/FA1 as a novel surface marker. Stem Cell Rev, 2009. 5(4): p. 353-68.
116. Sjogren-Jansson, E., et al., Large-scale propagation of four undifferentiated human embryonic stem cell lines in a feeder-free culture system. Dev Dyn, 2005. 233(4): p. 1304-14.
117. Ameen, C., et al., Human embryonic stem cells: current technologies and emerging industrial applications. Crit Rev Oncol Hematol, 2008. 65(1): p. 54-80.
118. Richards, M., et al., Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells. Nat Biotechnol, 2002. 20(9): p. 933-6.
119. Peiffer, I., et al., Use of xenofree matrices and molecularly-defined media to control human embryonic stem cell pluripotency: effect of low physiological TGF-beta concentrations. Stem Cells Dev, 2008. 17(3): p. 519-33.
120. Miyazaki, T., et al., Recombinant human laminin isoforms can support the undifferentiated growth of human embryonic stem cells. Biochem Biophys Res Commun, 2008. 375(1): p. 27-32.
121. Zhou, J., et al., mTOR supports long-term self-renewal and suppresses mesoderm and endoderm activities of human embryonic stem cells. Proc Natl Acad Sci U S A, 2009. 106(19): p. 7840-5.
122. Su, Z., et al., Differentiation of human embryonic stem cells into immunostimulatory dendritic cells under feeder-free culture conditions. Clin Cancer Res, 2008. 14(19): p. 6207-17.
123. Li, Z., et al., Feeder-free self-renewal of human embryonic stem cells in 3D porous natural polymer scaffolds. Biomaterials, 2010. 31(3): p. 404-12.
124. Braam, S.R., et al., Recombinant vitronectin is a functionally defined substrate that supports human embryonic stem cell self-renewal via alphavbeta5 integrin. Stem Cells, 2008. 26(9): p. 2257-65.
125. Rosler, E.S., et al., Long-term culture of human embryonic stem cells in feeder-free conditions. Dev Dyn, 2004. 229(2): p. 259-74.
126. Odell, I.D. and D. Cook, Immunofluorescence techniques. J Invest Dermatol, 2013. 133(1): p. e4.
127. A. Higuchi, Q.D. Ling, S. Kumar, M. Munusamy, A.A. Alarfajj, A. Umezawa, et al.
Design of polymeric materials for culturing human pluripotent stem cells: progress toward feeder-free and xeno-free culturing Prog Polym Sci, 39 (7) (2014), pp. 1348–1374
128. Takahashi, K. and S. Yamanaka, Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 2006. 126(4): p. 663-76.