Figure 6. Short- and long-term feeder-free culture on hit polymer arrays.

a, Efficiencies of various culture systems to support undifferentiated growth of dissociated hES cells. Two media conditions were used, labeled at the bottom: mEF-conditioned media (MEF-CM) or chemically defined media (mTeSR1). Several combinations of substrate and protein coating were used in conjunction with these media. Three substrates consisted of tissue culture polystyrene (TCPS), hit polymer 9 (“9”; see Figure 1a for monomer structure), and hit polymer 15A-30% (“15A”; see Figure 1a for monomer structures). Four protein coatings consisted of matrigel, bovine serum, human serum, and human vitronectin. Lastly, mEFs on gelatin-coated TCPS in regular hES media was also used. In each condition, efficiencies were calculated as the number of SSEA-4+ and Oct4+ colonies seen on day 7 normalized to the number of cells attached on day 1. This metric specifically reflects the ability of substrates to promote undifferentiated clonal cell growth after correcting for any differences in initial cell attachment. b, Immunostaining of dissociated hES cells propagated on hit FBS-coated “15A-30%” polymer for 7 days against Nanog (green) and Tra-1-60 (red), and on FBS-coated hit “9” polymer for 7 days against Oct4 (green) and SSEA-4 (red). Immunostaining of dissociated hiPS cells propagated on hit “15A-30%” polymer for 7 days; SSEA-4 (red) and cell nuclei (blue). For these studies, hiPS cells were immunostained against SSEA-4, and then the SSEA4+ sorted cell population was used. c, Karyotypic analysis of hES cells propagated on hit “9” polymer array for more than 2 months (>10 passages). A normal 46XY karyotype was maintained on the hit array. d, Gene expression analyses via RT-PCR of various differentiation markers for the three germ layers generated through embryoid body (EB) in vitro differentiation. Lane labels are as follows: “M-EB” for EBs generated from hES cells cultured on mEFs, “9-EB” and “15-EB” for EBs generated from hES cells cultured on 9 and 15A-30% hit polymer arrays respectively, and “9-ES” and “15-ES” for hES cells cultured on 9 and 15A-30% hit polymer arrays respectively. e, Teratoma formation in immunodeficient mice by cells cultured on “15A-30%” hit arrays. H&E staining was performed on the teratoma. Resulting teratoma contained tissues representing all three germ layers: ectoderm, epidermal and neural tissue (rosette); mesoderm, bone and cartilage; and endoderm, respiratory epithelium and intestinal-like epithelium. f, Fraction of adhered cells after 24 hr of culture in mTeSR1 media on hit polymer arrays coated with either human serum (HuSerum) or human vitronectin (HuVitronectin) and with the specified integrin blocking antibody. Cell number shown here are averages of 24 replicates of the following hit polymers: 15, 15B-10%, 15B-20%, 15B-25%, 15D-10, and 15D-20%. β₁ blocking had minimal effect either alone or in combination with α_vβ₅ and α_vβ₃ blocking, whereas both α_vβ₅ and α_vβ₃ blocking reduced adhesion.