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Analysis of mitochondrial function and localisation during human embryonic stem cell differentiation in vitro.

Prowse AB, Chong F, Elliott DA, Elefanty AG, Stanley EG, Gray PP, Munro TP, Osborne GW - PLoS ONE (2012)

Bottom Line: We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment.Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive.Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.

View Article: PubMed Central - PubMed

Affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, Australia. a.prowse@uq.edu.au

ABSTRACT
Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.

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Mitochondrial biogenesis agents enhance MIXL1 expression in differentiating hESC.(a) SNAP can induce MIXL1 expression in StemPro® 2D cultures independent of BMP4 addition (p<0.05, n = 4). (b)The pluripotency marker TG30 is down regulated in cells positive for mesendoderm marker MIXL1 at day3 post 250 µM SNAP treatment. (c) Differentiation to early mesoderm (day 3) is enhanced in 3D cultures by addition of mitochondrial biogenesis agents. Scale bars are 200 µM. (d) 250 µM SNAP can partially rescue MIXL1 expression on removal of Activin A or BMP4. Control samples were treated according to ([44], black bars) or cultured without BMP 4 (red bars) or without Activin A (blue bars). (e) Mitochondrially associated gene expression is highly variable after SNAP and AICAR treatment. S, SNAP; A, AICAR; M, metformin; POLG, polymerase gamma; TFAM, mitochondrial transcription factor A; numbers represent µM concentrations of reagents used; D, DMSO without biogenesis agents was added as controls in equal volumes to treated samples.
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pone-0052214-g001: Mitochondrial biogenesis agents enhance MIXL1 expression in differentiating hESC.(a) SNAP can induce MIXL1 expression in StemPro® 2D cultures independent of BMP4 addition (p<0.05, n = 4). (b)The pluripotency marker TG30 is down regulated in cells positive for mesendoderm marker MIXL1 at day3 post 250 µM SNAP treatment. (c) Differentiation to early mesoderm (day 3) is enhanced in 3D cultures by addition of mitochondrial biogenesis agents. Scale bars are 200 µM. (d) 250 µM SNAP can partially rescue MIXL1 expression on removal of Activin A or BMP4. Control samples were treated according to ([44], black bars) or cultured without BMP 4 (red bars) or without Activin A (blue bars). (e) Mitochondrially associated gene expression is highly variable after SNAP and AICAR treatment. S, SNAP; A, AICAR; M, metformin; POLG, polymerase gamma; TFAM, mitochondrial transcription factor A; numbers represent µM concentrations of reagents used; D, DMSO without biogenesis agents was added as controls in equal volumes to treated samples.

Mentions: Attenuation of mitochondrial function and promotion of glycolysis has been used to promote increased expression of pluripotency markers and inhibit differentiation [20]. Conversely, we sought to investigate whether promotion of mitochondrial biogenesis (and subsequently an increase in oxidative phosphorylation) would influence differentiation of hESC towards early mesoderm. We investigated three chemical agents, SNAP, AICAR and metformin with known effects on mitochondrial biogenesis and cell differentiation in human and other mammalian species [25], [38], [39], [40]. To determine if increasing mitochondrial biogenesis had any impact on differentiation, independent of factors to promote differentiation, MIXL1 cells were grown for 3–4 days on Geltrex coated plates in hESC maintenance media StemPro® with or without biogenesis agents. At day 4, 18.7±3.2% of cells treated with 250 µM SNAP were positive for MIXL1 expression (Figure 1a, p<0.05, n = 3, compared to untreated controls) and demonstrated down regulation of the pluripotency marker TG30 (Figure 1b) and SSEA-4 (not shown). Concentrations of SNAP at 250 µM or above had detrimental effects on cell number and mitochondrial membrane potential as assessed by JC-1 staining (Figure S1). Neither AICAR nor metformin increased the percentage of MIXL1 positive cells above untreated controls (Figure 1a). To determine if any biogenesis agents could increase MIXL1 positive cells during cardiogenic mesoderm induction, spin embryoid bodies were differentiated using APEL medium [34] and growth factors BMP4, Activin A, VEGF and SCF. Increasing concentrations of both SNAP and AICAR increased the percentage of MIXL1 positive cells 17.33±11.72 (p<0.05) and 13.41±13.4% (p>0.05) respectively above controls (Figure S2) as well as the relative level of MIXL1 expression within the cells (Figure 1c). In order to determine a positive impact of biogenesis agents on MIXL1 expression, embryoid bodies were formed in the presence of biogenesis agents diluted in DMSO with and without the key growth factors for differentiation, BMP4 and Activin A. As expected removal of either BMP4 or Activin A significantly impacted on MIXL1 expression (Figure 1d). However, MIXL1 expression was partially restored in cultures lacking BMP4 or Activin A by 250 µM SNAP, but not the DMSO control or AICAR treated samples (Figure 1d and S2 and 3). Mitochondrial biogenesis in hESC was measured by the expression of POLG and TFAM, nuclear encoded genes required for mitochondrial DNA replication and transcription respectively (for review see [11]). No treatment yielded a significant change in expression of POLG or TFAM (p>0.05). However both Metformin and the DMSO controls exhibited a trend in down regulation of each gene (Figure 1e). In contrast, SNAP and AICAR had a highly variable effect on gene expression and trended towards increasing expression of TFAM and POLG.


Analysis of mitochondrial function and localisation during human embryonic stem cell differentiation in vitro.

Prowse AB, Chong F, Elliott DA, Elefanty AG, Stanley EG, Gray PP, Munro TP, Osborne GW - PLoS ONE (2012)

Mitochondrial biogenesis agents enhance MIXL1 expression in differentiating hESC.(a) SNAP can induce MIXL1 expression in StemPro® 2D cultures independent of BMP4 addition (p<0.05, n = 4). (b)The pluripotency marker TG30 is down regulated in cells positive for mesendoderm marker MIXL1 at day3 post 250 µM SNAP treatment. (c) Differentiation to early mesoderm (day 3) is enhanced in 3D cultures by addition of mitochondrial biogenesis agents. Scale bars are 200 µM. (d) 250 µM SNAP can partially rescue MIXL1 expression on removal of Activin A or BMP4. Control samples were treated according to ([44], black bars) or cultured without BMP 4 (red bars) or without Activin A (blue bars). (e) Mitochondrially associated gene expression is highly variable after SNAP and AICAR treatment. S, SNAP; A, AICAR; M, metformin; POLG, polymerase gamma; TFAM, mitochondrial transcription factor A; numbers represent µM concentrations of reagents used; D, DMSO without biogenesis agents was added as controls in equal volumes to treated samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3526579&req=5

pone-0052214-g001: Mitochondrial biogenesis agents enhance MIXL1 expression in differentiating hESC.(a) SNAP can induce MIXL1 expression in StemPro® 2D cultures independent of BMP4 addition (p<0.05, n = 4). (b)The pluripotency marker TG30 is down regulated in cells positive for mesendoderm marker MIXL1 at day3 post 250 µM SNAP treatment. (c) Differentiation to early mesoderm (day 3) is enhanced in 3D cultures by addition of mitochondrial biogenesis agents. Scale bars are 200 µM. (d) 250 µM SNAP can partially rescue MIXL1 expression on removal of Activin A or BMP4. Control samples were treated according to ([44], black bars) or cultured without BMP 4 (red bars) or without Activin A (blue bars). (e) Mitochondrially associated gene expression is highly variable after SNAP and AICAR treatment. S, SNAP; A, AICAR; M, metformin; POLG, polymerase gamma; TFAM, mitochondrial transcription factor A; numbers represent µM concentrations of reagents used; D, DMSO without biogenesis agents was added as controls in equal volumes to treated samples.
Mentions: Attenuation of mitochondrial function and promotion of glycolysis has been used to promote increased expression of pluripotency markers and inhibit differentiation [20]. Conversely, we sought to investigate whether promotion of mitochondrial biogenesis (and subsequently an increase in oxidative phosphorylation) would influence differentiation of hESC towards early mesoderm. We investigated three chemical agents, SNAP, AICAR and metformin with known effects on mitochondrial biogenesis and cell differentiation in human and other mammalian species [25], [38], [39], [40]. To determine if increasing mitochondrial biogenesis had any impact on differentiation, independent of factors to promote differentiation, MIXL1 cells were grown for 3–4 days on Geltrex coated plates in hESC maintenance media StemPro® with or without biogenesis agents. At day 4, 18.7±3.2% of cells treated with 250 µM SNAP were positive for MIXL1 expression (Figure 1a, p<0.05, n = 3, compared to untreated controls) and demonstrated down regulation of the pluripotency marker TG30 (Figure 1b) and SSEA-4 (not shown). Concentrations of SNAP at 250 µM or above had detrimental effects on cell number and mitochondrial membrane potential as assessed by JC-1 staining (Figure S1). Neither AICAR nor metformin increased the percentage of MIXL1 positive cells above untreated controls (Figure 1a). To determine if any biogenesis agents could increase MIXL1 positive cells during cardiogenic mesoderm induction, spin embryoid bodies were differentiated using APEL medium [34] and growth factors BMP4, Activin A, VEGF and SCF. Increasing concentrations of both SNAP and AICAR increased the percentage of MIXL1 positive cells 17.33±11.72 (p<0.05) and 13.41±13.4% (p>0.05) respectively above controls (Figure S2) as well as the relative level of MIXL1 expression within the cells (Figure 1c). In order to determine a positive impact of biogenesis agents on MIXL1 expression, embryoid bodies were formed in the presence of biogenesis agents diluted in DMSO with and without the key growth factors for differentiation, BMP4 and Activin A. As expected removal of either BMP4 or Activin A significantly impacted on MIXL1 expression (Figure 1d). However, MIXL1 expression was partially restored in cultures lacking BMP4 or Activin A by 250 µM SNAP, but not the DMSO control or AICAR treated samples (Figure 1d and S2 and 3). Mitochondrial biogenesis in hESC was measured by the expression of POLG and TFAM, nuclear encoded genes required for mitochondrial DNA replication and transcription respectively (for review see [11]). No treatment yielded a significant change in expression of POLG or TFAM (p>0.05). However both Metformin and the DMSO controls exhibited a trend in down regulation of each gene (Figure 1e). In contrast, SNAP and AICAR had a highly variable effect on gene expression and trended towards increasing expression of TFAM and POLG.

Bottom Line: We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment.Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive.Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.

View Article: PubMed Central - PubMed

Affiliation: Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St. Lucia, Australia. a.prowse@uq.edu.au

ABSTRACT
Human embryonic stem cell (hESC) derivatives show promise as viable cell therapy options for multiple disorders in different tissues. Recent advances in stem cell biology have lead to the reliable production and detailed molecular characterisation of a range of cell-types. However, the role of mitochondria during differentiation has yet to be fully elucidated. Mitochondria mediate a cells response to altered energy requirements (e.g. cardiomyocyte contraction) and, as such, the mitochondrial phenotype is likely to change during the dynamic process of hESC differentiation. We demonstrate that manipulating mitochondrial biogenesis alters mesendoderm commitment. To investigate mitochondrial localisation during early lineage specification of hESCs we developed a mitochondrial reporter line, KMEL2, in which sequences encoding the green fluorescent protein (GFP) are targeted to the mitochondria. Differentiation of KMEL2 lines into the three germ layers showed that the mitochondria in these differentiated progeny are GFP positive. Therefore, KMEL2 hESCs facilitate the study of mitochondria in a range of cell types and, importantly, permit real-time analysis of mitochondria via the GFP tag.

Show MeSH
Related in: MedlinePlus