Lls from the neural lineage [42,43]. Embryoid bodies plated on laminin after 30 days of neural specific differentiation show GFP (through anti-GFP antibody binding) localisation to mitochondria in b-III-tubulin positive cells (Epigenetics Figure 4b-e) confirmed by co-staining with an antimitochondrial antibody (not shown). Further, mitochondrialPromotion of Oxidative Phosphorylation Enhances DifferentiationMitochondrial biogenesis is controlled by peroxisome proliferator-activated receptor-c coactivator-1a (PGC-1a), NRF-1 and TFAM [11]. Metformin and AICAR are known activators of AMP-activated protein kinase (AMPK) [39] which in turn increases the production of PGC-1a. PGC-1a co-activates theTracking Mitochondria during hESC Differentiationtranscription of TFAM [48], a direct regulator of mitochondrial DNA transcription and replication. SNAP is a nitric oxide (NO) donor, also known to increase expression of mitochondrial biogenesis genes such as TFAM and POLG however its mode of action is to directly activate PGC-1a [49] thus indirectly increasing mitochondrial biogenesis. The fold changes (1.5 to 3) we observed in the mitochondrial biogenesis regulators TFAM and POLG, although variable, concurred with published results [15,21,39,50]. In addition, SNAP and AICAR displayed a trend of increasing levels of TFAM and POLG suggesting increased mitochondrial biogenesis. We observed that SNAP induced mitochondrial biogenesis in cytokine free StemPro media lead to an increased production of 25837696 MIXL1+ cells. In contrast, neither Metformin nor AICAR induced expression in these conditions. Conversely, in differentiating embryoid bodies both SNAP and AICAR increased the number of MIXL1 positive cells by approximately 15 compared to untreated controls (Figure S2). Furthermore, in the absence of the key differentiation factors BMP4 or ACTIVIN A, SNAP was able to partially restore MIXL1 expression in embryoid bodies. However, AICAR could not substitute for these cytokines in the embryoid body assay. This suggests that SNAP and AICAR may have different modes of action in promoting differentiation. For example, SNAP may induce differentiation [38] through either mitochondrial biogenesis or an as yet Epigenetics unknown pathway, while AICAR may not induce differentiation but may inhibit pluripotency thereby improving the general differentiation of the cells regardless of lineage. A possible confounding factor is that embryoid bodies without BMP4 and ACTIVIN A were smaller compared to controls (Figure S3). Nevertheless, further testing of differentiation efficiency in combinatorial titrations of AICAR or SNAP in lineage specific differentiation protocols is needed to precisely define the role of mitochondria in differentiation.biogenesis (50 or 250uM) agents indicated or DMSO as control. Cells were grown feeder free on Geltrex coated plates. On day 3 cells were harvested and treated with 5uM JC-1 for 15mins at RT. Bars represent relative cell numbers with low membrane potential. 
Error bars are +/2SD of n = 3 biological replicates. S = SNAP, A = AICAR, M = Metformin. (PDF)Figure S2 MIXL1 expression post treatment with biogenesis agents. a) AICAR and SNAP at 500 mM in the presence of BMP4 and Activin A increase MIXL1 expression relative to controls. b) Individual replicate data represented in part “a” expressed as MIXL expression relative to control. c) Raw data of MIXL expression expressed as percentage positive for MIXL expression. n/a = test not performed, Dead = cell death proh.Lls from the neural lineage [42,43]. Embryoid bodies plated on laminin after 30 days of neural specific differentiation show GFP (through anti-GFP antibody binding) localisation to mitochondria in b-III-tubulin positive cells (Figure 4b-e) confirmed by co-staining with an antimitochondrial antibody (not shown). Further, mitochondrialPromotion of Oxidative Phosphorylation Enhances DifferentiationMitochondrial biogenesis is controlled by peroxisome proliferator-activated receptor-c coactivator-1a (PGC-1a), NRF-1 and TFAM [11]. Metformin and AICAR are known activators of AMP-activated protein kinase (AMPK) [39] which in turn increases the production of PGC-1a. PGC-1a co-activates theTracking Mitochondria during hESC Differentiationtranscription of TFAM [48], a direct regulator of mitochondrial DNA transcription and replication. SNAP is a nitric oxide (NO) donor, also known to increase expression of mitochondrial biogenesis genes such as TFAM and POLG however its mode of action is to directly activate PGC-1a [49] thus indirectly increasing mitochondrial biogenesis. The fold changes (1.5 to 3) we observed in the mitochondrial biogenesis regulators TFAM and POLG, although variable, concurred with published results [15,21,39,50]. In addition, SNAP and AICAR displayed a trend of increasing levels of TFAM and POLG suggesting increased mitochondrial biogenesis. We observed that SNAP induced mitochondrial biogenesis in cytokine free StemPro media lead to an increased production of 25837696 MIXL1+ cells. In contrast, neither Metformin nor AICAR induced expression in these conditions. Conversely, in differentiating embryoid bodies both SNAP and AICAR increased the number of MIXL1 positive cells by approximately 15 compared to untreated controls (Figure S2). Furthermore, in the absence of the key differentiation factors BMP4 or ACTIVIN A, SNAP was able to partially restore MIXL1 expression in embryoid bodies. However, AICAR could not substitute for these cytokines in the embryoid body assay. This suggests that SNAP and AICAR may have different modes of action in promoting differentiation. For example, 
SNAP may induce differentiation [38] through either mitochondrial biogenesis or an as yet unknown pathway, while AICAR may not induce differentiation but may inhibit pluripotency thereby improving the general differentiation of the cells regardless of lineage. A possible confounding factor is that embryoid bodies without BMP4 and ACTIVIN A were smaller compared to controls (Figure S3). Nevertheless, further testing of differentiation efficiency in combinatorial titrations of AICAR or SNAP in lineage specific differentiation protocols is needed to precisely define the role of mitochondria in differentiation.biogenesis (50 or 250uM) agents indicated or DMSO as control. Cells were grown feeder free on Geltrex coated plates. On day 3 cells were harvested and treated with 5uM JC-1 for 15mins at RT. Bars represent relative cell numbers with low membrane potential. Error bars are +/2SD of n = 3 biological replicates. S = SNAP, A = AICAR, M = Metformin. (PDF)Figure S2 MIXL1 expression post treatment with biogenesis agents. a) AICAR and SNAP at 500 mM in the presence of BMP4 and Activin A increase MIXL1 expression relative to controls. b) Individual replicate data represented in part “a” expressed as MIXL expression relative to control. c) Raw data of MIXL expression expressed as percentage positive for MIXL expression. n/a = test not performed, Dead = cell death proh.
