Figure 1.
Morphological changes of hMSCs feeder cells and H1 cells in media supplemented with 0, 0.4 or 4/ml bFGF.
Upper row: hMSCs feeder cells presented distinct morphologies in media supplemented with 0, 0.4 or 4 ng/ml bFGF. Bottom row: H1-4 ng culture differentiated into short spindle-shaped fibroblast like cells at P32+8 as indicated by the asterisk (*). See also Figure S1.
Figure 2.
H1, H9 and DF19 had higher growth rate in media supplemented with 0/ml vs. 4 ng/ml bFGF.
Cells that have been cultured with 0 or 0.4/ml bFGF supplement were split evenly into media with 0 or 4 ng/ml bFGF, 3 wells of each, and subsequently counted after 8 days of culture. The average percentage of cells in 4 ng/ml culture vs. 0 ng/ml was presented for each cell line (n = 3). Error bars represent standard deviation among triplicate repeats.
Figure 3.
Culture with 0/ml bFGF had increased number of colonies compared to culture with 4 ng/ml bFGF.
H9-4 ng, DF19-0 ng or DF19-4 ng culture was each split evenly 1∶6 into media supplemented with 0 or 4 ng/ml bFGF, 3 wells of each. Colonies were imaged and counted after 8 days of culture. The average number of colonies from triplicate wells was presented for each culture (n = 1). Error bars represent standard deviation among triplicates. Corresponding representative images were taken after ALP staining. *: P<0.01; **: P<0.1.
Figure 4.
H1 clonogenicity is inhibited by exogenous bFGF in a dosage dependent manner.
(a). Initial H1-0 ng culture was plated as single cells into 0 vs. 0.4 ng/ml bFGF, 0 vs. 4 ng/ml bFGF, or 0 vs. 4 ng/ml bFGF after 2 days (2D) of culture in 0 ng/ml bFGF media for clonogenicity comparison. (b). Representative images of ALP stained cultures as described in (a). (c). Left-hand three columns: Initial H1-0 ng, H1-0.4 ng and H1-4 ng culture were each plated as single cells into its corresponding culture condition for clonogenicity comparison. Right-hand three columns: Initial H1-0.4 ng culture was plated as single cells into 0, 0.4 and 4 ng/ml bFGF media for clonogenicity comparison. (d). Representative images of ALP stained cultures as described in (c), with top row images corresponding to the left-hand three columns in 4c and bottom row images corresponding to the right-hand three columns in 4c. The average number of colonies from triplicate wells was presented for each culture (n = 1) except for the right-hand three columns in 4c, which represent the average of duplicate wells (n = 2). Error bars represent standard deviation. *: P<0.01.
Figure 5.
H9 clonogenicity is inhibited by exogenous bFGF in a dosage dependent manner.
(a). Initial H9-0 ng or H9-0.4 ng/H9-4 ng culture was dissociated into single cells and plated into 0 vs. 0.04 vs. 0.4 vs. 4 ng/ml bFGF media, or 0 vs. 4 ng/ml bFGF media, respectively, at 5×104 cells/well. (b). Representative images of ALP stained cultures as described in (a). (c). Left-hand five columns: Initial H9-0.4 ng culture was dissociated into single cells and plated into 0 vs. 0.04 vs. 0.4 vs. 4 ng/ml bFGF or 4 ng/ml bFGF after 2 days (2D) of culture in 0 ng/ml bFGF media. Right-hand three columns: initial H9-0 ng, H9-0.4 ng and H9-4 ng culture were each plated as single cells into its corresponding culture condition, at 5×104 cells/well. (d). Representative images of ALP stained cultures as described in (c). The average number of colonies from triplicate wells was presented for each culture (n = 1). Error bars represent standard deviation. *: P<0.01; **: P<0.05; ***: P<0.1.
Figure 6.
DF19 clonogenicity is inhibited by exogenous bFGF in a dosage dependent manner.
(a) Initial DF19-0 ng was dissociated into single cells by accutase or trypLE and plated into 0 vs. 4 ng/ml bFGF media at 5×104 cells/well. The same experiment was carried out with initial DF19-4 ng cells using accutase. In addition, DF19-0 ng and DF19-4 ng were also plated as single cells into its corresponding 0 or 4 ng/ml bFGF media respectively. (b). Representative images of ALP stained cultures as described in (a). (c). Left-hand five columns: Initial DF19-0.4 ng culture was dissociated into single cells and plated into 0 vs. 0.04 vs. 0.4 vs. 4 ng/ml bFGF or 4 ng/ml bFGF after 2 days (2D) of culture in 0 ng/ml bFGF media. Right-hand three columns: initial DF19-0 ng, DF19-0.4 ng and DF19-4 ng culture were each plated as single cells into its corresponding culture condition, at 5×104 cells/well. (d). Representative images of ALP stained cultures as described in (c). The average number of colonies from triplicate wells was presented for each culture (n = 1). Error bars represent standard deviation. *: P<0.01; **: P = <0.05; ***: P = <0.1.
Figure 7.
Morphological differences in single cell cultures with 0 vs. 4/ml bFGF.
Top row: dividing cells were observed in 0 ng/ml bFGF culture but not in 4 ng/ml bFGF culture; Second row: degenerative colonies were observed in 4 ng/ml bFGF culture but not in 0 ng/ml culture; Third and fourth rows: Surviving colonies in 4 ng/ml bFGF culture were smaller and slender than in 0 ng/ml culture; Bottom row: Fibroblast-like differentiated cells (*) were observed in 4 ng/ml bFGF culture but not in 0 ng/ml culture, both of which were derived from H1-4 ng at P31+6. Scales in white: 50 µM; Scales in black: 200 µM.
Figure 8.
Exogenous bFGF promotes colony growth and inhibits differentiation of pluripotent stem cells cultured on MEF.
(a). ALP stained images of H1-MEF, H9-MEF and DF19-MEF cultured in 0 vs. 8 or 0 vs. 100 ng/ml bFGF media for 12 days. (b). H1-MEF-8 ng (P32) and DF19-MEF-100 ng (P33) single cells were plated at 2×105 cells/well into 0, 0.4 and 4 ng/ml bFGF media or 0, 4 and 100 ng/ml bFGF media, respectively. The average number of colonies from triplicate wells was presented for each culture (n = 1). Error bars represent standard deviation. (c). Representative images of ALP stained cultures as described in (b). (d). Top row: MEF feeder cells demonstrated similar morphological changes in response to different bFGF concentrations as shown by hMSCs feeder cells in Figure 1; Middle row: representative H1-MEF colonies in 0, 04 and 4 ng/ml bFGF media 4 days post initial plating; Bottom row: representative H1-MEF colonies in 0, 04 and 4 ng/ml bFGF media 8 days post initial plating.
Table 1.
H1, H9 and DF19 colony distribution in 0 vs. 8 or 0 vs. 100 ng/ml bFGF culture.
Figure 9.
Exogenous bFGF inhibits thawing efficiency in a dosage dependent manner across all three-cell lines.
Identical wells of H1-0 ng, H9-0 ng or DF19-0 ng culture were each frozen individually into a single vial after dissociation by collagenase or accutase. Subsequently each vial of frozen cells was plated equally into 0, 0.04, 0.4 or 4 ng/ml bFGF media, 3 wells of each. The average number of colonies from triplicate wells was presented for each culture (n = 2). Error bars represent standard deviation. Asterisks indicate statistical differences between different bFGF culture conditions in the accutased group. **: P<0.1; *: P<0.01. See also Figure S2.
Figure 10.
bFGF at 20 or 100/ml promoted DF19 differentiation in a dosage dependent manner but had limited effect on H9 cells.
Top two rows: Bright-field images and ALP stained images of H9-4 ng, H9-20 ng and H9-100 ng cultured in parallel at P40+11; Middle two rows: Bright-field images of feeder cell layers and colonies in DF19-4 ng, DF19-20 ng and DF19-100 ng cultures at P33+6, with the latter demonstrating more blurred boundaries between feeder cells and colonies in DF19-20 ng and DF19-100 ng; Bottom two rows: ALP stained images of DF19-4 ng, DF19-20 ng and DF19-100 ng cultures at P33+6. Scales in white: 50 µM; Scales in black: 200 µM.
Figure 11.
The effect of exogenous bFGF on H9 clonogenicity is similar at 4/ml or higher.
(a). Initial culture of H9-4 ng at P40+10 was dissociated into single cells by accutase and plated into 4, 20 or 100 ng/ml bFGF media at 5×104 cells/well, 2 wells of each, with or without 2 days of pre-incubation in 4 ng/ml bFGF media. Average number of colonies from duplicate wells was presented for each culture condition (n = 1). No statistically significant difference was observed between different culture conditions. (b). Representative ALP stained images of H9 culture in 4, 20 or 100 ng/ml bFGF media derived from H9-4 ng single cells.
Figure 12.
All cell lines cultured in 0, 0.4 or 4/ml bFGF media (excluding H1-4 ng) expressed both Oct 4 and SSEA4.
BF, bright field; DAPI, a blue fluorescent nucleic acid stain; Oct4, a pluripotent nuclear marker labeled by green fluorescence; SSEA4, a pluripotent cell surface marker labeled by red fluorescence; Oct4/SSEA4, overlay of both markers; DAPI/Oct4/SSEA4, overlay of all 3 markers.
Figure 13.
Flow cytometry analysis of SSEA1 and SSEA4 expression in all cell cultures.
A. H1, H9 and DF19 cells were analyzed after both 10/11 and 21/22 passages of culture on Ad-hMSCs feeder cells in 0, 0.4 or 4 ng/ml bFGF media. B. H9 and DF19 cells were analyzed after 13 and 8 passages of culture respectively on Ad-hMSCs feeder cells in 4, 20 or 100 ng/ml bFGF media. See also Figure S3.
Figure 14.
Expression of Oct4, Nanog and GDF3 was detected in all pluripotent cell cultures by RT-PCR.
Expression of HSP90, a house keeping gene, was detected in all samples including mitomycin C treated hMSCs feeder cells (MtC-hMSCs). Expression of Nanog, Oct4 and GDF3 were restricted to all pluripotent cell samples and not expressed in MtC-hMSCs (H1 and H9, n = 3; DF19, n = 2). Expression of all genes was negative in water and RNA control sample lanes (not shown). See also Figure S4.
Figure 15.
Expression of 3 germ lineage markers α-AFP, Nkx2.5 and Nestin was detected in EBs formed from all cell lines by RT-PCR.
Expression of HSP90, a housekeeping gene, was detected in all samples except for H9-0 ng at P25+14. Expression of α-AFP, Nkx2.5 and Nestin were detected in all EBs formed from all three cell lines except for H9-0 ng at P25+14, but not expressed in MtC-hMSCs (n = 3). Expression of all genes was negative in water and RNA control sample lanes (not shown).
Figure 16.
H1-0 ng at P32+19 gave rise to teratoma that contained all three germ layer lineage tissues.
Tumor: one kidney tumor (left) and one testis tumor (right) collected on day 19 post injection. Variously differentiated tissues from isolated tumors that represent the three germ layers are shown and indicated by arrows. Scale: 100 µM.
Figure 17.
Long-term cultured H1-0 ng and H9-0 ng cells had normal karyotype, whereas both DF19-0 ng and DF19-4 ng cells had abnormal karyotype characterized by trisomy 12.
Clonal aberration was observed in all cells examined in both DF19-0 ng and DF19-4 ng cultures at P29+16. Similar aberration was also observed in an independent culture of DF19-0 ng at P31+16, whereas the original culture of DF19 in TeSR1 media at P31 had normal karyotype.
Figure 18.
H1, H9 and DF19 demonstrated differential growth capacity in Ad-hMSCs conditioned media with or without bFGF supplement.
A. Images of H1 (P32+17+2) and H9 (P25+14+2) cultured in TeSR1, C0M (hESCs/hiPSCs media conditioned with MtC-hMSCs in the absence of bFGF) or (C0M+4 ng) (hESCs/hiPSCs media conditioned with MtC-hMSCs in the absence of bFGF and subsequently supplemented with 4 ng/ml bFGF). B. Flow cytometry analysis of SSEA1−SSEA4+ cells in H1 (P32+17+5), H9 (P25+14+5) and DF19 (P35+5) after 36–38 days of culture in TeSR1, C0M or (C0M+4 ng). C. Images of DF19 (P35+2) cultured in TeSR1, C0M, (C0M+4 ng) or C4M (hESCs/hiPSCs media conditioned with MtC-hMSCs in the presence of 4 ng/ml bFGF) on D1, D5 and D9 after plating. See also Figure S5.