HS-deficient mESCs cannot be maintained in the absence of serum during 2D expansion

HS-deficient EXT1-/- mESCs were maintained in three distinctly different 2D culture conditions. Of these, only two conditions were able to support EXT1-/- mESC expansion and viability long-term (> 10 passages). Serum-free conditions, although shown to previously support E14 mESCs during 2D culture, proved detrimental to EXT1-/- mESCs; mESCs displayed a rounded cell morphology, and became detatched from the culture dish (Figure 4.7 C). Quantification of viability confirmed this, since over 80 % of EXT1-/- mESCs grown in serum-free cultures were no longer viable by 48 h (Figure 4.8). In contrast, providing that serum was present (+/- feeder layer), EXT1-/- mESCs were maintained for more than 10 passages and displayed typical morphology, proliferation and colony formation (Figure 4.7 A and 4.7 B).

To further confirm that the EXT1-/- mESCs had not differentiated after 10 passages under the different culture conditions, co-immunostaining of Oct4 and Nanog was performed, common markers of ESC pluripotency. EXT1-/- mESCs remained positive for Oct4 and Nanog expression throughout expansion in both conditions (Figure 4.9). Quantification of Oct4 positive EXT1-/- mESCs confirmed that Oct4 expression was comparable to E14 mESCs, and additionally, there was no difference between culture conditions (+/- feeder layer). There was no difference in proportion of Nanog positive cells when comparing E14 mESCs and EXT1-/-

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mESCs cultured in the presence of serum and feeders (+F +FBS), however, in feeder-free conditions, HS-deficient EXT1-/- mESCs displayed higher proportions of Nanog positive cells compared to E14 ESCs cultured in the same condition. In addition, comparing the effects of culture condition on EXT1-/- mESCs pluripotency, Oct4 expression was unchanged, the proportion of Nanog-positive cells was however, significantly higher in EXT1-/- mESCs cultured in the absence of feeders, compared to EXT1-/- mESCs cultured with feeders (Figure 4.10).

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Figure 4.7 HS-deficient EXT1-/- mESC expansion was not supported in serum-free and feeder- free 2D culture conditions. EXT1-/- mESCs were maintained in vitro for more than 10 passages in three distinctly different culture conditions in parallel; with feeders and serum (+F + FBS), without feeders but with serum (–F + FBS) and without feeders or serum (–F –FBS), as explained previous. EXT1-/- mESC behaviour and morphology during monolayer culture varied depending on growth conditions. At 48 h EXT1-/- mESCs cultured in the presence of serum (A, B, D and E) (+F + FBS, –F + FBS) displayed typical behaviour; cells adhered to surface of dish and formed colonies. In contrast, EXT1-/- mESCs cultured in serum-free conditions (C and F) (-F –FBS) displayed abnormal behaviour; cells adopted a rounded morphology and became detatched from the dish surface, highlighted with arrows. Long term, the cells were unable to survive in serum-free, feeder-free conditions. Images are representative of the entire population for each condition and the experiment was repeated over 6 times. Scale bar represents 50 µm.

139 Time Try pa n bl ue + c e ll s (%) 0 20 40 60 80 100 120 EXT1-/- +F +FBS EXT1-/- -F +FBS EXT1-/- -F -FBS 0h 24h 48h 96h

Figure 4.9 Oct4 and Nanog expression in HS-deficient EXT1-/- mESCs cultured in the presence or absence of feeder cells. EXT1-/- mESCs were maintained in two different culture conditions in vitro for more than 10 passages, with and without a feeder layer. After 48 h post sub culture, the expression of Oct4 was comparable between the two culture conditions (B and F). However, Nanog positive cells appeared to be more abundant in feeder-free cultures (G) compared to cells cultured in the presence of feeders (C). Furthermore Oct4 and Nanog co-expressing cells (yellow cells labelled with arrows in C and G) appeared more abundant in EXT1-/- mESCs cultured in the absence of feeders (-F +FBS) (H) compared to cells cultured in the presence of feeders. Images are representative of the entire cell population and the experiment was repeated over 3 times. Scale bar represents 100 µm.

Figure 4.8 HS-deficient EXT1-/- mESC maintained in serum-free 2D conditions are not viable by 48 h. EXT1-/- mESCs were maintained in parallel in 3 different culture conditions; with feeders and serum (+F + FBS), without feeders but with serum (–F + FBS) and without feeders or serum (–F – FBS). The number of trypan blue- positive-cells were counted daily. After 96 h, EXT1-/- mESCs cultured in serum- free, feeder-free conditions were almost completely non-viable (99 %), demonstrating that this condition cannot support long term EXT1-/- mESC expansion. The experiment was repeated three times; error bars, SEM.

140 Condition p o si ti v e E S C s (% ) 0 20 40 60 80 100 120 Oct4 Nanog EXT1-/- ESC +F +FBS EXT1-/- ESC -F +FBS * E14 ESC +F +FBS E14 ESC -F +FBS *

Figure 4.10 The percentage of Nanog positive mESCs is higher in HS-deficient EXT1-/- mESCs compared to E14 mESCs but only in the absence of feeders. EXT1-/- mESCs and E14 mESCs were maintained in parallel for more than 10 passages with or without feeders but in the presence of serum. The proportion of Oct4 and Nanog positive EXT1-/- mESCs and E14 mESCs were comprable in +F +FBS conditions. However when cultured in the absence of feeders, EXT1-/- mESCs displayed significantly higher proportions of Nanog positive mESCs than E14 mESCs cultured in the same condition. EXT1-/- mESCs cultured in the absence of feeders displayed significantly higher proportions of Nanog positive cells when compared to EXT1-/- mESCs cultured with feeders. The proportion of Oct4 positive cells was not significantly different when comparing cell type and conditions. n = 4, * p < 0.05, student T-test, error bars represent SEM.

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