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February 16, 2013

Don’t remember, exactly, from where this little ditty of an article, came; believe someone other than I authored this. Interesting as hell, though, huh?

Metabolism is vital to every aspect of cell function, yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report, using an untargeted metabolomics approach, that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells, and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis, and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly, the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs, which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs, and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency, and in evaluating iPSC and ESC equivalence.
Keywords: reprogramming; iPS cells; metabolome; stem cells; metabolism
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of page
Introduction
Seminal advances in stem cell biology demonstrated that somatic cells could be reprogrammed back into a pluripotent state (termed induced pluripotent stem cells, or iPSCs) by expression of defined transcription factors1,2,3. Numerous studies have examined the genetic and epigenetic profiles of iPSCs, in relation to both their somatic cells, and to embryonic stem cells (ESCs), to gain insight into somatic cell reprogramming, as well as the quantitative and qualitative differences that may exist between these pluripotent cell types4,5. Yet, these important studies are not sufficient for generating a complete picture of the molecular components regulating cellular function.

Metabolism is either directly or indirectly involved with every aspect of cell function. Metabolomic technologies enable the examination and identification of endogenous biochemical reaction products, revealing information on the metabolic pathways and processes occurring within a living cell6,7,8. Here we examine the metabolomics of human iPSCs relative to ESCs and to their somatic cells of origin, which revealed the bioenergetic changes that take place in somatic cell reprogramming, and their function in regulating the reprogramming process.

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of page
Results
The metabolome profiles of iPSCs versus ESCs
To examine the metabolic profile associated with induced pluripotency, we used an untargeted metabolomics approach with liquid chromatography coupled to electrospray ionization quadrupole time-of-flight MS (LC-ESI-QTOF-MS)9 to analyze the relative abundance of metabolites in ESCs, iPSCs derived from human keratinocytes or fibroblasts, and their respective somatic cells of origin. All iPSCs used in this study (four fibroblast-derived iPSCs: FiPS4F1, FiPS4F2, FiPS4F4, FiPS4F5, and three keratinocyte-derived iPSCs: KiPS4FA, KiPS4FB and KiPS4F2) demonstrated pluripotent gene and protein expression, the ability to generate all three embryonic germ layers in vivo and a normal karyotype (Supplementary information, Data S1 and Figure S1)10,11,12. Our MS-based platform enabled us to observe greater than 5 000 metabolite features, defined as molecular entities with a unique mass/charge and retention time value. The relative abundance of metabolites for each cell population was then quantified by comparing the integrated area of each feature9.
Previous studies have demonstrated that iPSC gene expression profiles and methylation patterns are influenced by prolonged culture in vitro13,14,15. For example, iPSCs that have undergone a low number of passages retain aspects of epigenetic memory from their somatic source, which influences their differentiation potential14,15. Conversely, as iPSCs remain in culture and increase in passage number, they more closely genetically and functionally resemble ESCs13,15. Thus, we first examined the global metabolic profile of iPSCs at early and late passages compared to ESC controls. Importantly, since culture conditions can influence the metabolite compositions within cells, and variations can occur between cell lines, we compared identical iPSCs at early (p.16) and late (> p.41) passages that had been grown in chemically-defined medium (i.e. mTeSR1)16, to enable us to focus on metabolites that are more intrinsically distinct between cell types rather than culture-condition induced. While the global metabolic signature of early passage iPSCs was relatively close to ESC controls (5.3% difference), the metabolome signatures of late passage iPSCs grown in the same defined conditions was significantly closer to ESCs (0.23% difference). These data demonstrate that the metabolite profiles of iPSCs adapt to a more ESC-like state the longer they remain in culture (Figure 1).

Figure 1.

The metabolome profiles of iPSCs versus ESCs. Heat maps of metabolite features (> 5 000) in the indicated iPSC and ESC lines grown in chemically defined conditions at early (left panel) and late passage (right panel). The percentage of metabolite feature differences in iPSCs compared to ESCs is indicated below each respective heat map. Biological duplicates (e.g., (a) vs (b)) and experimental duplicates (e.g., (a) vs (a)) were performed.
Full figure and legend (68K)

Metabolic differences observed between iPSCs and ESCs reveal novel pathways important in somatic cell reprogramming
These findings are the first demonstrating that the global metabolomic profiles of iPSCs and ESCs are very similar, in concurrence with the overall genetic, epigenetic and functional similarities that have been reported comparing these pluripotent cell types4. Yet, metabolite differences do exist, and given the fact that recent studies have demonstrated that seemingly minor variations between iPSCs and ESCs may have significant phenotypic consequences17, we next identified metabolites that differed between these cell types. We identified eight metabolites that showed a greater than two-fold difference with a P-value < 0.01 between ESCs and late passage iPSCs. We had previously shown that high levels of

On Jan 6, 2013, at 7:28 PM, hofhaus4@comcast.net wrote:

Metabolism is vital to every aspect of cell function, yet the metabolome of induced pluripotent stem cells (iPSCs) remains largely unexplored. Here we report, using an untargeted metabolomics approach, that human iPSCs share a pluripotent metabolomic signature with embryonic stem cells (ESCs) that is distinct from their parental cells, and that is characterized by changes in metabolites involved in cellular respiration. Examination of cellular bioenergetics corroborated with our metabolomic analysis, and demonstrated that somatic cells convert from an oxidative state to a glycolytic state in pluripotency. Interestingly, the bioenergetics of various somatic cells correlated with their reprogramming efficiencies. We further identified metabolites that differ between iPSCs and ESCs, which revealed novel metabolic pathways that play a critical role in regulating somatic cell reprogramming. Our findings are the first to globally analyze the metabolome of iPSCs, and provide mechanistic insight into a new layer of regulation involved in inducing pluripotency, and in evaluating iPSC and ESC equivalence.

Keywords: reprogramming; iPS cells; metabolome; stem cells; metabolism
Top
of page
Introduction
Seminal advances in stem cell biology demonstrated that somatic cells could be reprogrammed back into a pluripotent state (termed induced pluripotent stem cells, or iPSCs) by expression of defined transcription factors1,2,3. Numerous studies have examined the genetic and epigenetic profiles of iPSCs, in relation to both their somatic cells, and to embryonic stem cells (ESCs), to gain insight into somatic cell reprogramming, as well as the quantitative and qualitative differences that may exist between these pluripotent cell types4,5. Yet, these important studies are not sufficient for generating a complete picture of the molecular components regulating cellular function.

Metabolism is either directly or indirectly involved with every aspect of cell function. Metabolomic technologies enable the examination and identification of endogenous biochemical reaction products, revealing information on the metabolic pathways and processes occurring within a living cell6,7,8. Here we examine the metabolomics of human iPSCs relative to ESCs and to their somatic cells of origin, which revealed the bioenergetic changes that take place in somatic cell reprogramming, and their function in regulating the reprogramming process.

Top
of page
Results
The metabolome profiles of iPSCs versus ESCs
To examine the metabolic profile associated with induced pluripotency, we used an untargeted metabolomics approach with liquid chromatography coupled to electrospray ionization quadrupole time-of-flight MS (LC-ESI-QTOF-MS)9 to analyze the relative abundance of metabolites in ESCs, iPSCs derived from human keratinocytes or fibroblasts, and their respective somatic cells of origin. All iPSCs used in this study (four fibroblast-derived iPSCs: FiPS4F1, FiPS4F2, FiPS4F4, FiPS4F5, and three keratinocyte-derived iPSCs: KiPS4FA, KiPS4FB and KiPS4F2) demonstrated pluripotent gene and protein expression, the ability to generate all three embryonic germ layers in vivo and a normal karyotype (Supplementary information, Data S1 and Figure S1)10,11,12. Our MS-based platform enabled us to observe greater than 5 000 metabolite features, defined as molecular entities with a unique mass/charge and retention time value.
The relative abundance of metabolites for each cell population was then quantified by comparing the integrated area of each feature9.
Previous studies have demonstrated that iPSC gene expression profiles and methylation patterns are influenced by prolonged culture in vitro13,14,15. For example, iPSCs that have undergone a low number of passages retain aspects of epigenetic memory from their somatic source, which influences their differentiation potential14,15. Conversely, as iPSCs remain in culture and increase in passage number, they more closely genetically and functionally resemble ESCs13,15. Thus, we first examined the global metabolic profile of iPSCs at early and late passages compared to ESC controls. Importantly, since culture conditions can influence the metabolite compositions within cells, and variations can occur between cell lines, we compared identical iPSCs at early (p.16) and late (> p.41) passages that had been grown in chemically-defined medium (i.e. mTeSR1)16, to enable us to focus on metabolites that are more intrinsically distinct between cell types rather than culture-condition induced. While the global metabolic signature of early passage iPSCs was relatively close to ESC controls (5.3% difference), the metabolome signatures of late passage iPSCs grown in the same defined conditions was significantly closer to ESCs (0.23% difference). These data demonstrate that the metabolite profiles of iPSCs adapt to a more ESC-like state the longer they remain in culture (Figure 1).

Figure 1.

The metabolome profiles of iPSCs versus ESCs. Heat maps of metabolite features (> 5 000) in the indicated iPSC and ESC lines grown in chemically defined conditions at early (left panel) and late passage (right panel). The percentage of metabolite feature differences in iPSCs compared to ESCs is indicated below each respective heat map. Biological duplicates (e.g., (a) vs (b)) and experimental duplicates (e.g., (a) vs (a)) were performed.
Full figure and legend (68K)

Metabolic differences observed between iPSCs and ESCs reveal novel pathways important in somatic cell reprogramming
These findings are the first demonstrating that the global metabolomic profiles of iPSCs and ESCs are very similar, in concurrence with the overall genetic, epigenetic and functional similarities that have been reported comparing these pluripotent cell types4. Yet, metabolite differences do exist, and given the fact that recent studies have demonstrated that seemingly minor variations between iPSCs and ESCs may have significant phenotypic consequences17, we next identified metabolites that differed between these cell types. We identified eight metabolites that showed a greater than two-fold difference with a P-value < 0.01 between ESCs and late passage iPSCs. We had previously shown that high levels of the rising tides lowered phenotypic dispositions.
Thank you.
Thank you very much.

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