Limits...
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.

Show MeSH

Related in: MedlinePlus

LDS-751 stains human embryonic stem cell mitochondria based on membrane potential.(a) LDS-751 is co-localised with GFP in the KMEL2 mitochondria reporter line and does not overlap with the nucleus. Fluorescence intensities for each fluorophore were measured along the 160 µm line shown in the overlay image and plotted as distance vs intensity. (b) Mitochondria specific staining is lost when treated with a mitochondrial membrane depolarising agent valinomycin. Line profile analysis demonstrates LDS-751 no longer localised to the mitochondria after blocking mitochondrial membrane potential. The line profile in the overlay image represents 140 µm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3526579&req=5

pone-0052214-g003: LDS-751 stains human embryonic stem cell mitochondria based on membrane potential.(a) LDS-751 is co-localised with GFP in the KMEL2 mitochondria reporter line and does not overlap with the nucleus. Fluorescence intensities for each fluorophore were measured along the 160 µm line shown in the overlay image and plotted as distance vs intensity. (b) Mitochondria specific staining is lost when treated with a mitochondrial membrane depolarising agent valinomycin. Line profile analysis demonstrates LDS-751 no longer localised to the mitochondria after blocking mitochondrial membrane potential. The line profile in the overlay image represents 140 µm.

Mentions: To further validate the use of KMEL2 in live tracking of hESC mitochondria, we used flow based image analysis to confirm mitochondrial GFP localisation. We initially used LDS-751 as a nuclear counter stain, because it has no significant spectral overlap with GFP. However, in LDS-751 stained KMEL2 cells, significant co-localisation of LDS-751 with GFP was observed (Figure S5). This suggests LDS-751 does not stain the nucleus in hESC. This was confirmed in a 2-dimensional format using DAPI as a nuclear stain. LDS-751 did not co-localise with the nuclear stain DAPI and, instead, LDS-751 overlapped exclusively with GFP imported to the mitochondria (Figure 3a). In addition, LDS-751 co-localised exclusively with mitosox red (Figure S5). Depolarisation of mitochondrial membranes with valinomyocin inhibited the localisation of LDS-751 to mitochondria (Figure 3b). Mitochondrial localisation of LDS-751 has previously been reported in mouse fibroblasts and monocytes and, as for hESC, was dependent on polarised mitochondrial membranes [41]. Thus, LDS-751 can be used as a tool for tracking mitochondria in cultured cells.


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)

LDS-751 stains human embryonic stem cell mitochondria based on membrane potential.(a) LDS-751 is co-localised with GFP in the KMEL2 mitochondria reporter line and does not overlap with the nucleus. Fluorescence intensities for each fluorophore were measured along the 160 µm line shown in the overlay image and plotted as distance vs intensity. (b) Mitochondria specific staining is lost when treated with a mitochondrial membrane depolarising agent valinomycin. Line profile analysis demonstrates LDS-751 no longer localised to the mitochondria after blocking mitochondrial membrane potential. The line profile in the overlay image represents 140 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0052214-g003: LDS-751 stains human embryonic stem cell mitochondria based on membrane potential.(a) LDS-751 is co-localised with GFP in the KMEL2 mitochondria reporter line and does not overlap with the nucleus. Fluorescence intensities for each fluorophore were measured along the 160 µm line shown in the overlay image and plotted as distance vs intensity. (b) Mitochondria specific staining is lost when treated with a mitochondrial membrane depolarising agent valinomycin. Line profile analysis demonstrates LDS-751 no longer localised to the mitochondria after blocking mitochondrial membrane potential. The line profile in the overlay image represents 140 µm.
Mentions: To further validate the use of KMEL2 in live tracking of hESC mitochondria, we used flow based image analysis to confirm mitochondrial GFP localisation. We initially used LDS-751 as a nuclear counter stain, because it has no significant spectral overlap with GFP. However, in LDS-751 stained KMEL2 cells, significant co-localisation of LDS-751 with GFP was observed (Figure S5). This suggests LDS-751 does not stain the nucleus in hESC. This was confirmed in a 2-dimensional format using DAPI as a nuclear stain. LDS-751 did not co-localise with the nuclear stain DAPI and, instead, LDS-751 overlapped exclusively with GFP imported to the mitochondria (Figure 3a). In addition, LDS-751 co-localised exclusively with mitosox red (Figure S5). Depolarisation of mitochondrial membranes with valinomyocin inhibited the localisation of LDS-751 to mitochondria (Figure 3b). Mitochondrial localisation of LDS-751 has previously been reported in mouse fibroblasts and monocytes and, as for hESC, was dependent on polarised mitochondrial membranes [41]. Thus, LDS-751 can be used as a tool for tracking mitochondria in cultured cells.

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