Serum-free derivation of human embryonic stem cell lines on human placental fibroblast feeders  Olga Genbacev, Ph.D., Ana Krtolica, Ph.D., Tamara Zdravkovic, M.S., Elisa Brunette, Ph.D., Sandra Powell, B.Sc., Aneel Nath, B.Sc., Eduardo Caceres, B.Sc., Michael McMaster, Ph.D., Susan McDonagh, Ph.D., Yan Li, Ph.D., Ramkumar Mandalam, Ph.D., Jane Lebkowski, Ph.D., Susan J. Fisher, Ph.D.  Fertility and Sterility  Volume 83, Issue 5, Pages 1517-1529 (May 2005) DOI: 10.1016/j.fertnstert.2005.01.086 Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 1 Characterization of three human placental fibroblast lines as candidate feeders for hESC propagation and derivation. (a) Growth characteristics of lines derived from individual placentas (7 weeks, n = 2; 8 weeks, n = 1). (b) Pathogen testing, which was conducted in two stages, showed that the 8B line was free of bacterial or viral DNA. (c) After X-irradiation, the 8B line continued to stain for vimentin (Vim), consistent with the mesenchymal origin of these cells. As expected, many of the fibroblasts had the morphology of senescent cells, a phenotype that is associated with X-irradiation. Fertility and Sterility 2005 83, 1517-1529DOI: (10.1016/j.fertnstert.2005.01.086) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 2 Placental fibroblasts support growth of the H9 hESC line in an undifferentiated state, as demonstrated by expression of markers of pluripotency. Morphologically, hESC colonies maintained for the same period of time on placental fibroblast (PLFb) feeders (right column) had a very similar appearance to those propagated on MEFs (left column). Under both conditions, the cells stained brightly for Oct-3/4, Tra 1-60, Tra 1-81, and SSEA-4. Alkaline phosphatase activity was also detected. Similar results were obtained with the H1 cell line (not shown). Fertility and Sterility 2005 83, 1517-1529DOI: (10.1016/j.fertnstert.2005.01.086) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 3 Derivation of new hESC lines on human placental fibroblast feeders. (a) After removal of the zona pellucida, an intact blastocyst was transferred in serum-free medium (knockout DMEM with KSR) to an irradiated placental fibroblast feeder layer (8B). By the next day, attachment was noted. Between weeks 2 and 3, a radial outgrowth of cells appeared. During this time trophectoderm cells (TE) capped the outgrowth. (b) After passaging seven times by manual dissection, the TE cells were lost and the embryonic cells (UCSF-1) grew as tightly packed, rounded colonies with typical hESC morphology. (c and d) At passage 9, UCSF-1 stained for Oct-3/4 and Tra 1-60. (e and f) At passage 4, UCSF-2 also stained for these markers of pluripotency. Fertility and Sterility 2005 83, 1517-1529DOI: (10.1016/j.fertnstert.2005.01.086) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 4 Characterization of the UCSF-1 hESC line that was maintained on human placental fibroblast feeders. (a) Cytogenetic analysis revealed that the UCSF-1 hESC line had a normal 46,XX karyotype. (b) Telomerase activity was measured at passage 7 by using the TRAP assay. No activity was observed in the buffer alone (Buff). As an additional negative control, a sample of 1,000 cell equivalents of each cell type was heat inactivated (Ht) and assayed in parallel. One thousand (1) and 5,000 (5) cell equivalents from 293 cells, a telomerase-positive E1 transformed cell line, served as a positive control. The same number of PLFb lacked demonstrable telomerase activity, which was detected in association with UCSF-1. However, the levels were lower than that in the control cells, since the hESCs were analyzed together with the feeders. (c) hTERT mRNA was analyzed by using quantitative real-time RT-PCR (Taqman). For comparison, three established hESC lines were analyzed in parallel: H1 at passage 32, H7 at passage 35, and H9 at passage 33. In all cases, these cells were maintained on growth factor–depleted Matrigel in MEF-conditioned medium. UCSF-1 was assayed after the cells were passaged 10 times by manual dissection or 16 times by collagenase dissociation. In both cases, UCSF-1 was cultured on 8B placental fibroblast feeders. As a negative control, the placental fibroblasts were assayed in parallel. (d) Samples from the same sources as shown in panel c were used to estimate Oct-3/4 mRNA levels by using quantitative RT-PCR (Taqman). (e) At passage 6, UCSF-1 hESCs were differentiated into embryoid bodies and analyzed by immunocytochemistry for the expression of markers of all three germ layers: α-fetoprotein (α-feto), endoderm; β-tubulin III (β-tub), ectoderm; and smooth muscle actin (m act), mesoderm. Fertility and Sterility 2005 83, 1517-1529DOI: (10.1016/j.fertnstert.2005.01.086) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions

FIGURE 5 UCSF-1 hESCs can be propagated under defined feeder-depleted conditions. (a) Appearance of UCSF-1 hESCs maintained for 11 passages on a laminin substrate in defined medium (X-VIVO 10) as viewed under a phase-contrast microscope. Note that dedifferentiated cells tended to fill in the spaces between the colonies. Under these culture conditions, the cells continued to express Oct-3/4. Nuclei were viewed by counterstaining with a DNA intercalating dye (DAPI). (b) TRAP analysis of UCSF-1 hESCs cultured under defined feeder-free conditions for 5 (p5) or 19 (p19) passages demonstrated robust telomerase activity. The assay was performed as described in the legend to Fig. 4 (see also Materials and Methods). (c and d) Real-time quantitative RT-PCR (Taqman) assays showed that UCSF-1 hESCs that were cultured for 14 passages under defined conditions (XVLam) expressed hTERT and Oct-3/4 mRNA levels that were comparable to those of H1 (passage 42), H7 (passage 27), and H9 hESCs cultured on growth factor–depleted Matrigel in MEF-conditioned medium. (e) After 32 weeks of culture on laminin in defined medium, passage 14 UCSF-1 cells were lifted from the matrix and allowed to differentiate into embryoid bodies. Immunolocalization detected expression markers of all three germ layers: α-feto, endoderm; β-tub, ectoderm; and m act, mesoderm. Fertility and Sterility 2005 83, 1517-1529DOI: (10.1016/j.fertnstert.2005.01.086) Copyright © 2005 American Society for Reproductive Medicine Terms and Conditions