Metabolic Remodeling during Early Cardiac Lineage Specification of Pluripotent Stem Cells.

Growing evidence indicates that metabolites and energy metabolism play an active rather than consequential role in regulating cellular fate. Cardiac development requires dramatic metabolic remodeling from relying primarily on glycolysis in pluripotent stem cells (PSCs) to oxidizing a wide array of energy substrates to match the high bioenergetic demands of continuous contraction in the developed heart. However, a detailed analysis of how remodeling of energy metabolism contributes to human cardiac development is lacking. Using dynamic multiple reaction monitoring metabolomics of central carbon metabolism, we evaluated temporal changes in energy metabolism during human PSC 3D cardiac lineage specification. Significant metabolic remodeling occurs during the complete differentiation, yet temporal analysis revealed that most changes occur during transitions from pluripotency to mesoderm (day 1) and mesoderm to early cardiac (day 5), with limited maturation of cardiac metabolism beyond day 5. Real-time metabolic analysis demonstrated that while hPSC cardiomyocytes (hPSC-CM) showed elevated rates of oxidative metabolism compared to PSCs, they still retained high glycolytic rates, confirming an immature metabolic phenotype. These observations support the opportunity to metabolically optimize the differentiation process to support lineage specification and maturation of hPSC-CMs.

Uncoupling of Proliferative Capacity from Developmental Stage During Directed Cardiac Differentiation of Pluripotent Stem Cells.

Lineage-specific differentiation of human-induced pluripotent stem cells (hiPSCs) into cardiomyocytes (CMs) offers a patient-specific model to dissect development and disease pathogenesis in a dish. However, challenges exist with this model system, such as the relative immaturity of iPSC-derived CMs, which evoke the question of whether this model faithfully recapitulates in vivo cardiac development. As in vivo cardiac developmental stage is intimately linked with the proliferative capacity (or maturation is inversely correlated to proliferative capacity), we sought to understand how proliferation is regulated during hiPSC CM differentiation and how it compares with in vivo mouse cardiac development. Using standard Chemically Defined Media 3 differentiation, gene expression profiles demonstrate that hiPSC-derived cardiomyocytes (hiPSC-CMs) do not progress past the equivalent of embryonic day 14.5 of murine cardiac development. Throughout differentiation, overall DNA synthesis rapidly declines with <5% of hiPSC-CMs actively synthesizing DNA at the end of the differentiation period despite their immaturity. Bivariate cell cycle analysis demonstrated that hiPSC-CMs have a cell cycle profile distinct from their non-cardiac counterparts from the same differentiation, with significantly fewer cells within G1 and a marked accumulation of cells in G2/M than their non-cardiac counterparts throughout differentiation. Pulse-chase analysis demonstrated that non-cardiac cells progressed completely through the cell cycle within a 24-h period, whereas hiPSC-CMs had restricted progression with only a small proportion of cells undergoing cytokinesis with the remainder stalling in late S-phase or G2/M. This cell cycle arrest phenotype is associated with abbreviated expression of cell cycle promoting genes compared with expression throughout murine embryonic cardiac development. In summary, directed differentiation of hiPSCs into CMs uncouples the developmental stage from cell cycle regulation compared with in vivo mouse cardiac development, leading to a premature exit of hiPSC-CMs from the cell cycle despite their relative immaturity.

Extracellular Flux Analysis of Mitochondrial Function in Pluripotent Stem Cells.

Mitochondrial function and energy metabolism are increasingly recognized not only as regulators of pluripotent stem cell function and fate, but also as critical targets in disease pathogenesis and aging. Therefore across the downstream applications of pluripotent stem cells, including development and disease modeling, drug screening, and cell-based therapies, it is crucial to be able to measure mitochondrial function and metabolism in a high-throughput, real-time and label-free manner. Here we describe the application of Seahorse extracellular flux analysis to measure mitochondrial function in pluripotent stem cells and their derivatives. Specifically, we highlight two assays, the Mitochondrial Stress Test, which quantifies overall mitochondrial function including basal, maximal and ATP-couple oxygen consumption rates, and the Electron Transport Chain Complex Specific assay, that quantifies function of individual complexes within the electron transport chain.

Energy Metabolism Regulates Stem Cell Pluripotency.

Pluripotent stem cells (PSCs) are characterized by their unique capacity for both unlimited self-renewal and their potential to differentiate to all cell lineages contained within the three primary germ layers. While once considered a distinct cellular state, it is becoming clear that pluripotency is in fact a continuum of cellular states, all capable of self-renewal and differentiation, yet with distinct metabolic, mitochondrial and epigenetic features dependent on gestational stage. In this review we focus on two of the most clearly defined states: "naïve" and "primed" PSCs. Like other rapidly dividing cells, PSCs have a high demand for anabolic precursors necessary to replicate their genome, cytoplasm and organelles, while concurrently consuming energy in the form of ATP. This requirement for both anabolic and catabolic processes sufficient to supply a highly adapted cell cycle in the context of reduced oxygen availability, distinguishes PSCs from their differentiated progeny. During early embryogenesis PSCs adapt their substrate preference to match the bioenergetic requirements of each specific developmental stage. This is reflected in different mitochondrial morphologies, membrane potentials, electron transport chain (ETC) compositions, and utilization of glycolysis. Additionally, metabolites produced in PSCs can directly influence epigenetic and transcriptional programs, which in turn can affect self-renewal characteristics. Thus, our understanding of the role of metabolism in PSC fate has expanded from anabolism and catabolism to include governance of the pluripotent epigenetic landscape. Understanding the roles of metabolism and the factors influencing metabolic pathways in naïve and primed pluripotent states provide a platform for understanding the drivers of cell fate during development. This review highlights the roles of the major metabolic pathways in the acquisition and maintenance of the different states of pluripotency.

Glycolytic Stimulation Is Not a Requirement for M2 Macrophage Differentiation.

Enhanced glucose uptake and a switch to glycolysis are key traits of M1 macrophages, whereas enhanced fatty acid oxidation and oxidative phosphorylation are the main metabolic characteristics of M2 macrophages. Recent studies challenge this traditional view, indicating that glycolysis may also be critically important for M2 macrophage differentiation, based on experiments with 2-DG. Here we confirm the inhibitory effect of 2-DG on glycolysis, but also demonstrate that 2-DG impairs oxidative phosphorylation and significantly reduces 13C-labeled Krebs cycle metabolites and intracellular ATP levels. These metabolic derangements were associated with reduced JAK-STAT6 pathway activity and M2 differentiation marker expression. While glucose deprivation and glucose substitution with galactose effectively suppressed glycolytic activity, there was no effective suppression of oxidative phosphorylation, intracellular ATP levels, STAT6 phosphorylation, and M2 differentiation marker expression. These data indicate that glycolytic stimulation is not required for M2 macrophage differentiation as long as oxidative phosphorylation remains active.

Interferon Gamma Induces Reversible Metabolic Reprogramming of M1 Macrophages to Sustain Cell Viability and Pro-Inflammatory Activity.

Classical activation of M1 macrophages with lipopolysaccharide (LPS) is associated with a metabolic switch from oxidative phosphorylation to glycolysis. However, the generalizability of such metabolic remodeling to other modes of M1 macrophage stimulation, e.g. type II interferons (IFNs) such as IFNγ, has remained unknown as has the functional significance of aerobic glycolysis during macrophage activation. Here we demonstrate that IFNγ induces a rapid activation of aerobic glycolysis followed by a reduction in oxidative phosphorylation in M1 macrophages. Elevated glycolytic flux sustains cell viability and inflammatory activity, while limiting reliance on mitochondrial oxidative metabolism. Adenosine triphosphate (ATP) distributed by aerobic glycolysis is critical for sustaining IFN-γ triggered JAK (Janus tyrosine kinase)-STAT-1 (Signal Transducer and Activator of Transcription 1) signaling with phosphorylation of the transcription factor STAT-1 as its signature trait. Inhibition of aerobic glycolysis not only blocks the M1 phenotype and pro-inflammatory cytokine/chemokine production in murine macrophages and also human monocytes/macrophages. These findings extend on the potential functional role of immuno-metabolism from LPS- to IFNγ-linked diseases such as atherosclerosis and autoimmune disease.

Age-Related Accumulation of Somatic Mitochondrial DNA Mutations in Adult-Derived Human iPSCs.

The genetic integrity of iPSCs is an important consideration for therapeutic application. In this study, we examine the accumulation of somatic mitochondrial genome (mtDNA) mutations in skin fibroblasts, blood, and iPSCs derived from young and elderly subjects (24-72 years). We found that pooled skin and blood mtDNA contained low heteroplasmic point mutations, but a panel of ten individual iPSC lines from each tissue or clonally expanded fibroblasts carried an elevated load of heteroplasmic or homoplasmic mutations, suggesting that somatic mutations randomly arise within individual cells but are not detectable in whole tissues. The frequency of mtDNA defects in iPSCs increased with age, and many mutations were non-synonymous or resided in RNA coding genes and thus can lead to respiratory defects. Our results highlight a need to monitor mtDNA mutations in iPSCs, especially those generated from older patients, and to examine the metabolic status of iPSCs destined for clinical applications.

Energy metabolism in the acquisition and maintenance of stemness.

Energy metabolism is traditionally considered a reactive homeostatic system addressing stage-specific cellular energy needs. There is however growing appreciation of metabolic pathways in the active control of vital cell functions. Case in point, the stem cell lifecycle--from maintenance and acquisition of stemness to lineage commitment and specification--is increasingly recognized as a metabolism-dependent process. Indeed, metabolic reprogramming is an early contributor to the orchestrated departure from or reacquisition of stemness. Recent advances in metabolomics have helped decipher the identity and dynamics of metabolic fluxes implicated in fueling cell fate choices by regulating the epigenetic and transcriptional identity of a cell. Metabolic cues, internal and/or external to the stem cell niche, facilitate progenitor pool restitution, long-term tissue renewal or ensure adoption of cytoprotective behavior. Convergence of energy metabolism with stem cell fate regulation opens a new avenue in understanding primordial developmental biology principles with future applications in regenerative medicine practice.

1α,25-Dihydroxyvitamin D3 Regulates Mitochondrial Oxygen Consumption and Dynamics in Human Skeletal Muscle Cells.

Muscle weakness and myopathy are observed in vitamin D deficiency and chronic renal failure, where concentrations of the active vitamin D3 metabolite, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3), are low. To evaluate the mechanism of action of 1α,25(OH)2D3 in skeletal muscle, we examined mitochondrial oxygen consumption, dynamics, and biogenesis and changes in expression of nuclear genes encoding mitochondrial proteins in human skeletal muscle cells following treatment with 1α,25(OH)2D3. The mitochondrial oxygen consumption rate (OCR) increased in 1α,25(OH)2D3-treated cells. Vitamin D3 metabolites lacking a 1α-hydroxyl group (vitamin D3, 25-hydroxyvitamin D3, and 24R,25-dihydroxyvitamin D3) decreased or failed to increase OCR. 1α-Hydroxyvitamin D3 did not increase OCR. In 1α,25(OH)2D3-treated cells, mitochondrial volume and branching and expression of the pro-fusion protein OPA1 (optic atrophy 1) increased, whereas expression of the pro-fission proteins Fis1 (fission 1) and Drp1 (dynamin 1-like) decreased. Phosphorylated pyruvate dehydrogenase (PDH) (Ser-293) and PDH kinase 4 (PDK4) decreased in 1α,25(OH)2D3-treated cells. There was a trend to increased PDH activity in 1α,25(OH)2D3-treated cells (p = 0.09). 83 nuclear mRNAs encoding mitochondrial proteins were changed following 1α,25(OH)2D3 treatment; notably, PDK4 mRNA decreased, and PDP2 mRNA increased. MYC, MAPK13, and EPAS1 mRNAs, which encode proteins that regulate mitochondrial biogenesis, were increased following 1α,25(OH)2D3 treatment. Vitamin D receptor-dependent changes in the expression of 1947 mRNAs encoding proteins involved in muscle contraction, focal adhesion, integrin, JAK/STAT, MAPK, growth factor, and p53 signaling pathways were observed following 1α,25(OH)2D3 treatment. Five micro-RNAs were induced or repressed by 1α,25(OH)2D3. 1α,25(OH)2D3 regulates mitochondrial function, dynamics, and enzyme function, which are likely to influence muscle strength.

Metabolic rescue in pluripotent cells from patients with mtDNA disease.

Mitochondria have a major role in energy production via oxidative phosphorylation, which is dependent on the expression of critical genes encoded by mitochondrial (mt)DNA. Mutations in mtDNA can cause fatal or severely debilitating disorders with limited treatment options. Clinical manifestations vary based on mutation type and heteroplasmy (that is, the relative levels of mutant and wild-type mtDNA within each cell). Here we generated genetically corrected pluripotent stem cells (PSCs) from patients with mtDNA disease. Multiple induced pluripotent stem (iPS) cell lines were derived from patients with common heteroplasmic mutations including 3243A>G, causing mitochondrial encephalomyopathy and stroke-like episodes (MELAS), and 8993T>G and 13513G>A, implicated in Leigh syndrome. Isogenic MELAS and Leigh syndrome iPS cell lines were generated containing exclusively wild-type or mutant mtDNA through spontaneous segregation of heteroplasmic mtDNA in proliferating fibroblasts. Furthermore, somatic cell nuclear transfer (SCNT) enabled replacement of mutant mtDNA from homoplasmic 8993T>G fibroblasts to generate corrected Leigh-NT1 PSCs. Although Leigh-NT1 PSCs contained donor oocyte wild-type mtDNA (human haplotype D4a) that differed from Leigh syndrome patient haplotype (F1a) at a total of 47 nucleotide sites, Leigh-NT1 cells displayed transcriptomic profiles similar to those in embryo-derived PSCs carrying wild-type mtDNA, indicative of normal nuclear-to-mitochondrial interactions. Moreover, genetically rescued patient PSCs displayed normal metabolic function compared to impaired oxygen consumption and ATP production observed in mutant cells. We conclude that both reprogramming approaches offer complementary strategies for derivation of PSCs containing exclusively wild-type mtDNA, through spontaneous segregation of heteroplasmic mtDNA in individual iPS cell lines or mitochondrial replacement by SCNT in homoplasmic mtDNA-based disease.

Metabolic determinants of embryonic development and stem cell fate.

Decoding stem cell metabolism has implicated a tight linkage between energy metabolism and cell fate regulation, a dynamic interplay vital in the execution of developmental and differentiation programs. The inherent plasticity in energy metabolism enables prioritisation of metabolic pathways in support of stage-specific demands. Beyond traditional support of energetic needs, intermediate metabolism may also dictate cell fate choices through regulation of cellular signalling and epigenetic regulation of gene expression. The notion of a 'metabolism-centric' control of stem cell differentiation has been informed by developmental embryogenesis based upon an on-demand paradigm paramount in defining diverse developmental behaviours, from a post-fertilisation nascent zygote to complex organogenesis leading to adequate tissue formation and maturation. Monitored through natural or bioengineered stem cell surrogates, nutrient-responsive metabolites are identified as mediators of cross-talk between metabolic flux, cell signalling and epigenetic regulation charting, collectively, whether a cell will self-renew to maintain progenitor pools, lineage specify to ensure tissue (re)generation or remain quiescent to curb stress damage. Thus, bioenergetics are increasingly recognised as integral in governing stemness and associated organogenic decisions, paving the way for metabolism-defined targets in control of embryology, stem cell biology and tissue regeneration.

Loss of HSulf-1 promotes altered lipid metabolism in ovarian cancer.

BACKGROUND: Loss of the endosulfatase HSulf-1 is common in ovarian cancer, upregulates heparin binding growth factor signaling and potentiates tumorigenesis and angiogenesis. However, metabolic differences between isogenic cells with and without HSulf-1 have not been characterized upon HSulf-1 suppression in vitro. Since growth factor signaling is closely tied to metabolic alterations, we determined the extent to which HSulf-1 loss affects cancer cell metabolism. RESULTS: Ingenuity pathway analysis of gene expression in HSulf-1 shRNA-silenced cells (Sh1 and Sh2 cells) compared to non-targeted control shRNA cells (NTC cells) and subsequent Kyoto Encyclopedia of Genes and Genomics (KEGG) database analysis showed altered metabolic pathways with changes in the lipid metabolism as one of the major pathways altered inSh1 and 2 cells. Untargeted global metabolomic profiling in these isogenic cell lines identified approximately 338 metabolites using GC/MS and LC/MS/MS platforms. Knockdown of HSulf-1 in OV202 cells induced significant changes in 156 metabolites associated with several metabolic pathways including amino acid, lipids, and nucleotides. Loss of HSulf-1 promoted overall fatty acid synthesis leading to enhance the metabolite levels of long chain, branched, and essential fatty acids along with sphingolipids. Furthermore, HSulf-1 loss induced the expression of lipogenic genes including FASN, SREBF1, PPARγ, and PLA2G3 stimulated lipid droplet accumulation. Conversely, re-expression of HSulf-1 in Sh1 cells reduced the lipid droplet formation. Additionally, HSulf-1 also enhanced CPT1A and fatty acid oxidation and augmented the protein expression of key lipolytic enzymes such as MAGL, DAGLA, HSL, and ASCL1. Overall, these findings suggest that loss of HSulf-1 by concomitantly enhancing fatty acid synthesis and oxidation confers a lipogenic phenotype leading to the metabolic alterations associated with the progression of ovarian cancer. CONCLUSIONS: Taken together, these findings demonstrate that loss of HSulf-1 potentially contributes to the metabolic alterations associated with the progression of ovarian pathogenesis, specifically impacting the lipogenic phenotype of ovarian cancer cells that can be therapeutically targeted.

Stem cell lineage specification: you become what you eat.

Nutrient availability and intermediate metabolism are increasingly recognized to govern stem cell behavior. Oburoglu et al. (2014) now demonstrate that glutamine- and glucose-dependent nucleotide synthesis segregate erythroid versus myeloid differentiation during hematopoietic stem cell specification, implicating a metabolism-centric regulation of lineage choices.

Metabolic regulation of redox status in stem cells.

SIGNIFICANCE: Metabolism-dependent generation of reactive oxygen species (ROS) and associated oxidative damage have been traditionally linked to impaired homeostasis and cellular death. Beyond the adverse effects of ROS accumulation, increasing evidence implicates redox status as a regulator of vital cellular processes. RECENT ADVANCES: Emerging studies on the molecular mechanisms guiding stem cell fate decisions indicate a role for energy metabolism in regulating the fundamental ability of maintaining stemness versus undergoing lineage-specific differentiation. Stem cells have evolved protective metabolic phenotypes to minimize reactive oxygen generation through oxidative metabolism and support antioxidant scavenging through glycolysis and the pentose phosphate pathway. CRITICAL ISSUES: While the dynamics in ROS generation has been correlated with stem cell function, the intimate mechanisms by which energy metabolism regulates ROS to impact cellular fate remain to be deciphered. FUTURE DIRECTIONS: Decoding the linkage between nutrient sensing, energy metabolism, and ROS in regulating cell fate decisions would offer a redox-dependent strategy to regulate stemness and lineage specification.

Erratum to: Loss of HSulf-1 promotes altered lipid metabolism in ovarian cancer.

[This corrects the article DOI: 10.1186/2049-3002-2-13.].

Stem cell systems informatics for advanced clinical biodiagnostics: tracing molecular signatures from bench to bedside.

Development of innovative high throughput technologies has enabled a variety of molecular landscapes to be interrogated with an unprecedented degree of detail. Emergence of next generation nucleotide sequencing methods, advanced proteomic techniques, and metabolic profiling approaches continue to produce a wealth of biological data that captures molecular frameworks underlying phenotype. The advent of these novel technologies has significant translational applications, as investigators can now explore molecular underpinnings of developmental states with a high degree of resolution. Application of these leading-edge techniques to patient samples has been successfully used to unmask nuanced molecular details of disease vs healthy tissue, which may provide novel targets for palliative intervention. To enhance such approaches, concomitant development of algorithms to reprogram differentiated cells in order to recapitulate pluripotent capacity offers a distinct advantage to advancing diagnostic methodology. Bioinformatic deconvolution of several "-omic" layers extracted from reprogrammed patient cells, could, in principle, provide a means by which the evolution of individual pathology can be developmentally monitored. Significant logistic challenges face current implementation of this novel paradigm of patient treatment and care, however, several of these limitations have been successfully addressed through continuous development of cutting edge in silico archiving and processing methods. Comprehensive elucidation of genomic, transcriptomic, proteomic, and metabolomic networks that define normal and pathological states, in combination with reprogrammed patient cells are thus poised to become high value resources in modern diagnosis and prognosis of patient disease.

Disease-causing mitochondrial heteroplasmy segregated within induced pluripotent stem cell clones derived from a patient with MELAS.

Mitochondrial diseases display pathological phenotypes according to the mixture of mutant versus wild-type mitochondrial DNA (mtDNA), known as heteroplasmy. We herein examined the impact of nuclear reprogramming and clonal isolation of induced pluripotent stem cells (iPSC) on mitochondrial heteroplasmy. Patient-derived dermal fibroblasts with a prototypical mitochondrial deficiency diagnosed as mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) demonstrated mitochondrial dysfunction with reduced oxidative reserve due to heteroplasmy at position G13513A in the ND5 subunit of complex I. Bioengineered iPSC clones acquired pluripotency with multilineage differentiation capacity and demonstrated reduction in mitochondrial density and oxygen consumption distinguishing them from the somatic source. Consistent with the cellular mosaicism of the original patient-derived fibroblasts, the MELAS-iPSC clones contained a similar range of mtDNA heteroplasmy of the disease-causing mutation with identical profiles in the remaining mtDNA. High-heteroplasmy iPSC clones were used to demonstrate that extended stem cell passaging was sufficient to purge mutant mtDNA, resulting in isogenic iPSC subclones with various degrees of disease-causing genotypes. On comparative differentiation of iPSC clones, improved cardiogenic yield was associated with iPSC clones containing lower heteroplasmy compared with isogenic clones with high heteroplasmy. Thus, mtDNA heteroplasmic segregation within patient-derived stem cell lines enables direct comparison of genotype/phenotype relationships in progenitor cells and lineage-restricted progeny, and indicates that cell fate decisions are regulated as a function of mtDNA mutation load. The novel nuclear reprogramming-based model system introduces a disease-in-a-dish tool to examine the impact of mutant genotypes for MELAS patients in bioengineered tissues and a cellular probe for molecular features of individual mitochondrial diseases.

Lipid metabolism greases the stem cell engine.

Metabolic plasticity is increasingly postulated to be vital in the transition between stemness maintenance and lineage specification. Knobloch et al. (2012) now demonstrate that regulation of lipogenesis by fatty acid synthase and Spot14-dependent malonyl-CoA supply determines the proliferative activity of resident neural stem cells, contributing to adult neurogenesis.

Nuclear reprogramming with c-Myc potentiates glycolytic capacity of derived induced pluripotent stem cells.

Reprogramming strategies influence the differentiation capacity of derived induced pluripotent stem (iPS) cells. Removal of the reprogramming factor c-Myc reduces tumorigenic incidence and increases cardiogenic potential of iPS cells. c-Myc is a regulator of energy metabolism, yet the impact on metabolic reprogramming underlying pluripotent induction is unknown. Here, mitochondrial and metabolic interrogation of iPS cells derived with (4F) and without (3F) c-Myc demonstrated that nuclear reprogramming consistently reverted mitochondria to embryonic-like immature structures. Metabolomic profiling segregated derived iPS cells from the parental somatic source based on the attained pluripotency-associated glycolytic phenotype and discriminated between 3F versus 4F clones based upon glycolytic intermediates. Real-time flux analysis demonstrated a greater glycolytic capacity in 4F iPS cells, in the setting of equivalent oxidative capacity to 3F iPS cells. Thus, inclusion of c-Myc potentiates the pluripotent glycolytic behavior of derived iPS cells, supporting c-Myc-free reprogramming as a strategy to facilitate oxidative metabolism-dependent lineage engagement.