Figure 1.
Rest+/− and Rest−/− AB-1 ESCs cultured in the absence of feeder cells show impaired self-renewal that is restored upon prolonged culture.
(A, B) Alkaline phosphatase based self-renewal assay shows that the self-renewal efficiency of the N9 (Rest+/−) and N8 (Rest−/−) ESCs was significantly lower than that of the parental AB-1 ESCs at passage 2 but not at passage 10. (A) quantification of self-renewal assays. (B) representative pictures of colonies from self-renewal assays. The numbers under the lines show p-values (n = 3). Error bars are standard error of means. Cells were cultured in absence of feeder cells and in presence of LIF. (C) Venn diagram showing comparison of genes that showed a significant change in expression during the transition between passage 2 and passage 10 by genome-wide expression profiling. N9 (Rest+/−) and N8 (Rest−/−) cells showed significant changes in gene expression (N9 ∼965 and N8 ∼1296) compared to WT cells (∼65). (D, E, F and G) Heat map showing comparison of 50 genes with the highest fold changes that were either (D and F) up-regulated or (E and G) down-regulated in Rest+/− and Rest−/− cells during the transition from passage 2 to passage 10 to the expression of these genes in ES and differentiating cells based on FunGenES database.
Table 1.
Comparison of gene expression levels for N9 (Rest+/−)/N6 WT and N8 (Rest−/−)/N6 (WT) cultured on gelatin for passage 2.
Table 2.
Comparison of expression levels of genes for WT (N6), N9 (Rest+/−) and N8 (Rest−/−) during the transition between passage 2 and passage 10 when cultured on gelatin.
Figure 2.
Restoration of the impaired self-renewal and pluripotency of Rest+/− and Rest−/− AB-1 ESCs upon prolonged culture correlates, at least in part, with increased ESC pluripotency pathways.
(A, B and C) Ingenuity pathway analysis comparing canonical pathways in N6 (WT) cells during the transition between passage 2 and passage 10. While the wild-type cells did not have any significant changes in cellular pathways (except three, A), several pathways including the human embryonic stem cell pluripotency in Rest+/− (B) and Nanog embryonic stem cell pluripotency pathway changed in Rest−/− cells (C). (D, E and F) Validation of the results of genome-wide analyses in Rest+/− and Rest−/− ESCs. (D) Western blotting assays to measure REST protein levels (left panel: Western blot, right panels: quantification of western blot) and the core pluripotency factors Oct4, Sox2, and Nanog (left panel: Representative Western blot, right panels: quantification of three western blots for Rest+/− and four western blots for Rest−/−) in cells cultured for 2, 5, and 10 passages. Actin was used as a loading control. Error bars are standard error of means. Quantification of western blots was performed using Odyssey V3.0 software and was plotted after normalization against actin. REST protein levels were elevated in Rest+/− cells over passages but, it remained unchanged in the wild-type and Rest−/− cells. Whereas the wild-type cells maintained high levels of the core self-renewal factors Oct4, Sox2, and Nanog throughout the passages, their levels were significantly lower during the early passages of Rest +/− and Rest−/− cells and increased with passage. Nanog levels increased the most in Rest−/− cells. (E) Relative expression levels of Rest in N9 (Rest+/−) and N8 (Rest−/−) cells (confirmed with three independent primer sets) are shown after normalization with N6 (WT) cells grown for equivalent passage. Passage numbers are shown on the x-axis. p-values are shown over the bars. Gapdh was used as control. Error bars are standard error of means (n = 3). (F) Relative expression levels of pluripotency factors (Nanog, Oct4 and Sox2) in N9 (Rest+/−) and N8 (Rest−/−) cells are shown after normalization with N6 (WT) cells grown for equivalent passages. Passage numbers are shown on the x-axis. The p-values are shown over the bars. Gapdh was used as control. Error bars are standard error of means (n = 3).
Figure 3.
Extracellular cues provided by feeder cells compensate for REST-mediated loss of self-renewal and pluripotency in AB-1 ESCs.
(A) Alkaline phosphatase based self-renewal assays of AB-1 parental, Rest+/− and Rest−/− ESCs in the presence of LIF but in the presence or absence of feeder cells shows that Rest+/− and Rest−/− cells have lower self-renewal efficiency compared to the parental line in the absence of feeder cells, but no large difference in the presence of feeder cells. The numbers under the lines show p-values (n = 3). Error bars are standard error of means. (B) Representative pictures of AP-stained colonies of parental and Rest−/− cells in the presence of feeder layer. (C) Western blotting assays to detect level of REST and the core self-renewal regulators Oct4, Sox2, and Nanog (left panel: Western blot, right panels: quantification of western blot) confirmed that the feeder cells compensated for the loss of self-renewal and pluripotency in Rest+/− and Rest−/− AB-1 ESCs. Experiments were performed as described in Figure 2D.
Figure 4.
Extracellular cues provided by feeder cells compensate for REST-mediated loss of self-renewal and pluripotency in E14Tg2a.4 ESCs.
(A,B) Rest Mediated pluripotency is influenced by extracellular environment in E14Tg2a.4 ESCs. (A) MEF feeder cells and (B) the extracellular matrix component, laminin, countered lowered self-renewal in E14Tg2a.4 ESCs treated with siRNA mediated knockdown of REST (siRest). Percentages of self-renewing colonies in ES, NT and siRest treated cells after alkaline phosphatase assay are shown for each conditions (as labeled on x-axis). Both MEF and laminin condition resulted in maintenance of self-renewal compared to gelatin condition after siRest treatment. Number above bars are p-values (n = 3). Error bars are standard error of means. (C) Laminin maintains the expression of pluripotent markers in siRest treated E14Tg2a.4 ESCs. ESCs transfected with siRest or NT were plated on gelatin- or laminin-coated surfaces and were analyzed by QRT-PCR assays to determine the expression levels of Sox2, Nanog, and Oct4 transcripts. Number above bars are p-values (n = 3). Error bars are standard error of means. (D,E) Laminin counters lowered self-renewal and pluripotency due to loss of REST in shRest-mediated stable knock-down of REST in ESCs. (D) shRest lowered self-renewal when cells were cultured on gelatin but not on laminin. Percentages of self-renewing colonies in shRest and NT cells are shown for each conditions (as labeled on x-axis). Number above bars are p-values (n = 3). Error bars are standard error of means. (E) Stable shRest lines, when compared to shNT lines, of ESCs showed decreased levels of REST proteins when the cells were cultured either on gelatin or on laminin; however, the levels of Sox2, Nanog, and Oct4 were decreased only when cells were cultured on gelatin but not laminin.