Stem Cell Lineage Specification: You Become What You Eat

Abstract

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 oflineage choices.

Flexibility in energy metabolism enables cells to prioritize metabolic pathways in order to support stage-specific energetic demands (Folmes et al., 2012). Interrogation of stem cell metabolism has identified glycolys is as a key player in the maintenance of stemness through provision of energy and anabolic precursors (Folmes et al., 2011), while oxidative metabolism allows for more efficient energy production to match energy demanding processes of differentiating progeny(Chung et al., 2007). Furthermore, individual metabolic pathways underlying stem cell renewal versus lineage specification are starting to be elucidated. For instance, PPARδ-dependent fatty acid oxidation supports hematopoietic stem cell (HSC) pool maintenance through asymmetrical cell division, with inhibition of this pathway leading to HSC exhaustion due to symmetrical cell division into differentiated progenitors (Ito et al., 2012). Regulation of fatty acid oxidation versus synthesis may thus represent a rheostat of stem cell fate (Folmes et al., 2013), exemplified by the requirement for fatty acid synthesis in proliferating neural stem and progenitor cells(Knobloch et al., 2013). However, metabolic pathways that drive stem cells along parallel lineage paths remain to be defined in line with metabolite-mediated changes in the epigenetic state that prime stem cells to undergo fate conversions(Shyh-Chang et al., 2013a). A study published in Cell Stem Cell now identifies glutamine and glucose-dependent nucleotide biosynthesis as critical for murine and human HSC lineage specification there by extending the metabolic blueprint of stem cell identity and fate (Oburoglu et al., 2014).

Mammalian cells predominantly utilize glucose and glutamine as substrates to produce energy and precursors for biosynthetic reactions. However, the glucose transporter GLUT1 is not expressed in hematopoietic progenitors and only up regulated during late stages of human erythropoiesis, suggesting that alternative metabolic pathways may regulate HSC lineage commitment. Oburoglu et al. (2014) showed that HSCs and their progenitors express the ASCT2 glutamine transporter, and found that knock down of ASCT2 during erythropoietin (EPO)-induced differentiation skewed lineage commitment of HSCs away from the erythroid lineage (Figure 1A) in favor of the myeloid lineage (Figure 1B). In contrast, knockdown of GLUT1 did not impact lineage distribution. As ASCT2 can transport other amino acids in addition to glutamine, the authors confirmed that inhibition of glutaminolys is by 6-diazo-5-oxo-L-norleucine(DON) directed lineage commitment to the myeloid fate, while inhibition of glucose utilization by 2-deoxyglucose (2-DG) promoted the erythroid fate (Figure 1). Compensated by intracellular glutamine synthesis, removal of extracellular glutamine had little effect on erythropoiesis. Impairing glutaminolys is attenuated the stimulating effect of 2-DG on erythroid commitment, indicating the dominant nature of glutamine catabolism.

Figure 1. Glutamine and glucose metabolism regulates hematopoietic stem cell lineage specification.

Regulation of glutamine and glucose metabolism defines the ability of hematopoietic stem cells (HSC) to differentiate into erythroid versus myeloid lineages in vitro and in vivo. A) Glutamine uptake and glutaminolys is in support of de novo nucleotide biosynthesis and tricarboxylic acid cycle α-ketoglutarate generation are critical for HSC erythroid lineage commitment. Alternative metabolic pathways that support nucleotide synthesis, including 2-deoxyglucose (2-DG) blunting of glucose metabolism away from glycolys is toward the pentose phosphate pathway promotes erythropoiesis. B) In contrast, impairing nucleotide biosynthesis during erythropoietin-induced differentiation by impeding upstream glutamine synthesis (methionine sulfoximine, MSO), glutamine uptake, glutaminolys is (6-diao-5-oxo-L-norleucine, DON), transaminase reactions (aminooxyacetic acid, AOA) or blocking glucose entry into the pentose phosphate pathway (6-aminonicotinamide, 6-AN) skews lineage commitment away from erythroid, and toward myeloid fate. Supplementation with nucleosides rescues erythropoiesis, establishing the importance of de novo nucleotide biosynthesis for erythroid lineage commitment. GDH – glutamine dehydrogenase, GPAT – glutamine phosphoribosylpyrophosphateamidotransferase, GS – glutamine synthetase, G-6-P – glucose-6-phosphate, G6PD –glucose-6-phosphate dehydrogenase, PRA 5-Phosphoribosylamine, PRPP– 5-phosphoribosyl-1-pyrophosphate.

Using simultaneous tracing of glutamine carbons and nitrogens, the authors identified specific glutamine-dependent metabolic pathways critical for erythrogenes is, including a transaminase-dependent increase in alpha-ketoglutarate, as well as a stimulation of de novo purine and pyrimidine nucleotide synthesis in response to EPO-induced erythropoiesis(Oburoglu et al., 2014). Blocking transaminases and nucleotide synthesis impaired erythropoiesis, a defect rescued by a cell-permeable ester of alpha-ketoglutarate and nucleosides supplementation, respectively. Concomitant blockade of both pathways was only rescued by replenishing nucleosides, establishing the prevailing role of glutamine-dependent nucleotide biosynthesis for erythroid commitment (Figure 1A). In addition, the pentose phosphate pathway, which is elevated in EPO-treated cells and augmented by 2-DG treatment, may drive nucleotide biosynthesis and represents, a potential mechanism by which 2-DG promotes erythroid commitment (Figure 1A).

To determine the impact of glucose and glutamine metabolism in vivo, Oburoglu and colleagues used a mouse model of hemolytic anemia induced by phenyl hydrazine injection. Within five days of induction, control and 2-DG treated mice showed rebound erythropoiesis, which was attenuated by blocking glutaminolys is with DON(Oburoglu et al., 2014). Sustained anemia in DON-treated mice was specific to erythrogenes is as markers of ongoing HSC differentiation (i.e., Gr1⁺ cells) were significantly elevated with little effect on distribution of bone marrow progenitors. The effect of blocking glutamine metabolism was evident during stress, as treatment of nonanemic mice with DON did not impact progenitor distribution. In newborn mice, which display active hematopoietic lineage commitment, DON treatment reduced the number of Ter119⁺ erythroid progenitors and, within this population, attenuated erythroid commitment yet increased Gr1⁺ cells, while 2-DG treatment reduced myelomonocytic lineages. Thus, the authors demonstrated that opposing regulatory roles of glucose versus glutamine metabolism on erythroid and myelomonocytic cell fate extend in vivo.

Nucleotide biosynthesis, regardless of its origin, appears therefore critical for erythropoiesis (Figure 1), yet additional studies are required to delineate whether this observation matches a higher anabolic demand or whether these pathways interact with epigenetic regulators to drive cell fate. The ability to metabolically guide lineage commitment provides insight into the regulation of stem cell fate, opening new avenues for investigation. For example, can metabolic pathways offer targets in guiding discrete fate choices during lineage specification? What is the timing of these events and how do these pathways interact with genetics/epigenetics to regulate cell fate? What is the impact of metabolic regulation on stem cell physiology and pathophysiology? Growing evidence implicates that metabolic changes occur early during transitions between cellular fates (Folmes et al., 2011), which may act by epigenetically priming cells to support their ultimate fate (Shyh-Chang et al., 2013a). Consistent with this concept, glutaminolys is appears critical during early erythroid commitment though dispensable once this lineage is established(Oburoglu et al., 2014). It is intriguing that modulating energy metabolism may be exploited to facilitate efficient progenitor generation for regenerative applications and that nutrient changes in the niche can impact stem cell function in health and disease. Recent evidence indicates that reprogramming cellular metabolism can enhance the regenerative capacity of adult tissue. Case in point, there activation of Lin28a expression, which wanes during fetal development and aging, enhances adult tissue repair by accelerating glycolys is and oxidative metabolism (Shyh-Chang et al., 2013b). Indeed, inhibition of oxidative phosphorylation abrogates the benefits of Lin28a, while induction of oxidative phosphorylation augments repair capacity. This implicates that the decline in regenerative capacity with aging may be a result of compromised cellular metabolism leading to impaired stem cell function, a concept supported by evidence that caloric restriction can enhance skeletal muscle stem cell function by promoting oxidative metabolism (Cerletti et al., 2012). Modulating energy metabolism may thus offer therapeutic inroads for regenerative applications in aging and disease.

Linking discrete metabolic pathways to differentiation of distinct lineages opens the prospect of charting a metabolic map underlying stem cell differentiation. Building upon initial observations, future studies are needed to identify metabolite signals that influence lineage specification and on resolving the mechanisms by which metabolic pathways can interact with (epi)genetic regulators to control cell fate. In turn, modulation of energy metabolism may serve for targeted derivation of progenitor populations suitable for regenerative therapies, and for promoting resident pool function to ensure tissue self-repair.