Feeder-free

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 19 971-974... 

Efficiency of stably integrated shRNA vectors should be confirmed in vitro. Therefore, expand two positives clones per shRNA construct on feeders, freeze aliquots, and cultivate the cells feeder-free on gelatine-coated 10 cm dishes for at least 2 days. Harvest cells and analyze the four clones for knockdown efficiency as described in steps 2 and 3 of Subheading 3.1.2. Determine the best performing clone for each siRNA target sequence. 

Maintenance ofhPSC Maintain hPSC (H9, H13, H14, and 19-9-11) on mouse embryonic feeder cells in hESC medium DMEM/ F12 culture medium supplemented with 20 % KOSR, 0.1 mM NEAA, 1 mM L-glutamine, 0.1 mM 3ME, and 10 ng/ml human bFGF. For feeder-free culture, maintain the hPSC on matrigel in the presence of mTeSRl medium. 

Carpenter M K, Rosier E S, Fisk G J, et al. (2004). Properties of four human embryonic stem cell lines maintained in a feeder-free culture system. Dev. Dyn. 229 243-258. 

Van Hoof D, Braam S R, Dormeyer W, Ward-van Oostwaard D, Heck A J, Krijgsveld J and Mummery C L (2008), Feeder-free monolayer cultures of human embryonic stem cells express an epithelial plasma membrane protein profile . Stem Cells, 26,2777-81. 

Kang, M. and Han, Y.-M. (2014) Differentiation of human pluripotent stem cells into nephron progenitor cells in a serum and feeder free system. PLoS One 9, e94888. 

Biomaterial Design for Human ESCs and iPSCs on Feeder-Free Culture toward Pharmaceutical... 

Figure 6.1 Culture methods for human pluripotent stem cells (hPSCs). hPSCs can be cultured on mouse embryonic fibroblasts (MEFs) or human feeder cells. Feeder-free culturing of hPSCs is possible on xeno-containing Matrigel. Several types of feeder-free and xeno-free cultures of hPSCs can be developed on extracellular matrix (ECM)-coated surfaces and ohgopeptide-immobilLzed surfaces. hPSCs can also be cultured on polysaccharide hydrogels or on synthetic polymer surfaces by selecting a specific GAG or a specific polymer with optimal water content. Reproduced with permission from [2] Copyright 2014 Elsevier Inc.

Table 6.1 Feeder-free culture of hPSCs on ECM-immobilized substrates in a defined medium. Reproduced with permission from [2]. Copyright 2014 Elsevier Inc.

Medical engineering scientists and molecular biologists have recently examined the effects of culture conditions on stem cell fate. Additionally, since 2010, several novel biomaterials and methods for hPSC culture on these biomaterials have been developed under feeder-free and xeno-free culture conditions. Therefore, this chapter mainly focuses on the current developments in hPSC culture materials and describes the biomaterial-assisted regulation of hPSCs xmder feeder-free and xeno-free culture conditions. 

Natural polymers of recombinant or natural fibronectin, laminin, vitronectin, and collagen IV, which are components of ECMs, have begun to be used instead of Matrigel or decellularized ECMs for the feeder-free growth of undifferentiated hPSCs because their chemical characteristics are relatively more well defined (Table 6.1). The ECM components that are immobilized on dishes for hPSC proliferation and to provide binding sites for stem cells are summarized in Table 6.4 [50]. 

Figure 6.11 Chemical structures of the synthetic polymers used as substrates, hydrogels, and scaffolds for the proliferation of pluripotent hPSCs in defined media under feeder-free and xeno-free conditions.

A. Higuchi, Q.D. Ling, Y.A. Ko, Y. Chang, and A. Umezawa, Biomaterials for the feeder-free culture of human embryonic stem cells and induced pluripotent stem cells, Chem. Rev., Ill (5) 3021-3035, May. 2011. 

S. T. Hsu, and G.J. Wu, Preparation of induced pluripotent stem cells on dishes grafted on oligopeptide under feeder-free conditions, J. Taiwan Inst. Chem. Eng., 45 (2) 295-301, Mar. 2014. 

P.C. Beltrao-Braga, G.C. Pignatari, PC. Maiorka, N.A. Oliveira, N.F. Lizier, C.V. Wenceslau, M.A. Miglino, A.R. Muotri, and 1. Kerkis, Feeder-free derivation of induced pluripotent stem cells from human immature dental pulp stem cells. Cell Transplant., 20 (11-12) 1707-1719, Nov. 2011. 

H. Nandivada, L.G. ViUa-Diaz, K.S. O Shea, G.D. Smith, P.H. Krebsbach, and J. Lahann, Fabrication of synthetic polymer coatings and their use in feeder-free culture of human embryonic stem cells. Nature Protoc., 6 (7) 1037-1043, Jun. 2011. 

D. Ilic, E. Stephenson, V. Wood, L. Jacquet, D. Stevenson, A. Petrova, N. Kadeva, S. Codognotto, H. Patel, M. Semple, G. Cornwell, C. Ogilvie, and P. Braude, Derivation and feeder-free propagation of human embryonic stem cells under xeno-free conditions,... 

D. Hernandez, L. Ruban, and C. Mason, Feeder-free culture of human embryonic stem cells for scalable expansion in a reproducible manner. Stem Cells Develop., 20 (6) 1089-1098, Jun. 2011. 

T.M. Yoon, B. Chang, H.T. Kim, J.H. Jee, D.W. Kim, and D.Y Hwang, Human embryonic stem cells (hESCs) cultured under distinctive feeder-free culture conditions display global gene expression patterns similar to hESCs from feeder-dependent culture conditions. Stem Cell Rev. 6 (3) 425-437, Sep. 2010. 

Z. Li, M. Leung, R. Hopper, R. Ellenbogen, and M. Zhang, Feeder-free self-renewal of human embryonic stem cells in 3D porous natural polymer scaffolds. Biomaterials, 31 (3) 404-412, Jan. 2010. 

N. Siti-Ismail, A.E. Bishop, J.M. Polak, and A. Mantalaris, The benefit of human embryonic stem cell encapsulation for prolonged feeder-free maintenance. Biomaterials, 29 (29) 3946-3952, Oct. 2008. 

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