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Keywords:

  • Stem cell;
  • Proliferation;
  • Differentiation;
  • Omega-3 fatty acids;
  • Omega-6 fatty acids;
  • Eicosanoids;
  • Stem cell therapy

Abstract

  1. Top of page
  2. Abstract
  3. Current Challenges in Stem Cell Therapy
  4. Essential Fatty Acid Metabolism and Cell Proliferation and Differentiation
  5. Essential Fatty Acids and Stem Cell Proliferation
  6. Essential Fatty Acids and Stem Cell Differentiation
  7. Perspectives
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References

Stem cell therapy holds great promise for regenerative medicine and the treatment of numerous diseases. A key issue of stem cell therapy is the control of stem cell fate, but safe and practical methods are limited. Essential fatty acids, namely ω-6 (n-6) and ω-3 (n-3) polyunsaturated fatty acids (PUFA), and their metabolites are critical components of cell structure and function, and could therefore influence stem cell fate. The available evidence demonstrates that n-6 and n-3 PUFA and their metabolites can act through multiple mechanisms to promote the proliferation and differentiation of various stem cell types. Therefore, elucidating the role of PUFA and their metabolites in stem cell fate regulation is both a challenge and an opportunity for stem cell biology as well as stem cell therapy. PUFA-based interventions to create a favorable environment for stem cell proliferation or differentiation may thus be a promising and practical approach to controlling stem cell fate for clinical applications. Stem Cells 2014;32:1092–1098


Current Challenges in Stem Cell Therapy

  1. Top of page
  2. Abstract
  3. Current Challenges in Stem Cell Therapy
  4. Essential Fatty Acid Metabolism and Cell Proliferation and Differentiation
  5. Essential Fatty Acids and Stem Cell Proliferation
  6. Essential Fatty Acids and Stem Cell Differentiation
  7. Perspectives
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References

Recent advances in stem cell research have generated much excitement over their potential therapeutic applications. The key feature of stem cells, including embryonic stem cells (ESCs), adult stem cells, and induced pluripotent stem cells (iPSCs), is their ability to differentiate into specialized cell types and self-renew to produce more stem cells [1-3]. Stem cells can be introduced into organs or tissues to replace diseased or damaged cells with minimal risk of rejection and side effects, and can therefore be used to treat a number of diseases, including Alzheimer's disease, spinal cord injury, stroke, heart disease, diabetes, and cancer [4, 5]. The critical factor for the success of stem cell therapy is the capability to control stem cell fate. The development of safe, effective, and practical means to manipulate stem cell proliferation and differentiation is therefore the priority of the field.

Research has so far elucidated a number of signaling pathways and factors involved in stem cell proliferation and differentiation, such as extrinsic stimuli, intracellular signaling molecules, nuclear receptors, transcription factors, and chromatin remodeling [6-9]. This knowledge has enabled us to induce stem cell renewal and differentiation into target cell types as well as reprogram somatic cells back into iPSCs [5]. Currently, common approaches to manipulating stem cell fate include genetic modification, by over-expressing and/or knocking-down genes or pathways that are involved in stem cell differentiation or pluripotency [10, 11], and administration of biochemical cocktails composed of inductive factors, typically peptide and protein-based molecules such as growth factors, hormones, and cytokines [12]. However, these methods are not wholly practical for in vivo applications. Thus, new research efforts have been devoted to identifying small chemical molecules to formulate functional cocktails that can modulate stem cell fate, as an alternative to genetic modification. Many nutrients, such as vitamins, minerals, and PUFA and their metabolites, are important for embryonic and organ development, but their potential role in stem cell fate regulation has not been fully explored. Recent studies have demonstrated that certain vitamins and minerals, such as vitamin B3, C, D, folic acid, selenium, and retinoic acid, are involved in promoting the proliferation and differentiation of stem cells [13-16]. It has also been revealed that essential fatty acids and their metabolites may possess stem cell fate-modulating properties. This review will hereby focus on the role of essential fatty acids and their metabolites in the regulation of stem cell proliferation and differentiation.

Essential Fatty Acid Metabolism and Cell Proliferation and Differentiation

  1. Top of page
  2. Abstract
  3. Current Challenges in Stem Cell Therapy
  4. Essential Fatty Acid Metabolism and Cell Proliferation and Differentiation
  5. Essential Fatty Acids and Stem Cell Proliferation
  6. Essential Fatty Acids and Stem Cell Differentiation
  7. Perspectives
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References

Lipids have long been considered to be the building blocks of cellular membranes and adipose tissues as well as energy substrates. However, increasing evidence indicates that lipids, particularly the ω-6 (n-6) and ω-3 (n-3) PUFA, play significant roles in cell signaling and gene expression, and are thereby linked to many physiological and pathological processes.

The n-6 linoleic acid (LA; 18:2n-6) and n-3 α-linolenic acid (ALA; 18:3n-3) are essential fatty acids, as they cannot be synthesized de novo in mammals and must therefore be obtained from the diet [17]. Omega-6 and n-3 PUFA are not interconvertible [17]. LA and ALA share the same metabolic enzymes, and are converted through a series of desaturation and elongation reactions into their respective 20-carbon products: arachidonic acid (AA; 20:4n-6) and eicosapentaenoic acid (EPA; 20:5n-3) (Fig. 1). AA and EPA serve as the primary precursors for the synthesis of lipid mediators, including prostaglandins (PG), leukotrienes (LT), thromboxanes (TX), resolvins (Rv), protectins, and epoxyeicosatrienoic acids, through three major pathways: cyclooxygenase (COX), lipoxygenase, and cytochrome P450 (Fig. 1). Another important n-3 PUFA, docosahexaenoic acid (DHA, 22:6n-3), can be derived from EPA or from the diet, and is a major component of the brain and nervous system that can be metabolized to form key lipid mediators, such as Rv and protectins [18]. Lipid mediators derived from n-6 and n-3 PUFA are metabolically distinct and often have opposing physiological and pathological functions; for example, n-6 PUFA-derived eicosanoids tend to promote inflammation, while n-3 PUFA-derived lipid mediators largely inhibit inflammation [19].

image

Figure 1. The metabolic pathways for n-6 PUFA and n-3 PUFA. COX, LOX, and cytochrome P450 convert n-6 arachidonic acid and n-3 EPA into eicosanoids, including PG, LT, TX, and HETE. The n-3 DHA can be converted into Rv and PD. Abbreviations: ALA, α-linolenic acid; COX, cyclooxygenases; DHA, docosahexaenoic acid; DGLA, dihomo-gamma-linolenic acid; EPA, eicosapentaenoic acid; ETA, eicosatetraenoic acid; GLA, gamma-linolenic acid; HETE, hydroxyeicosatetraenoic acids; LA, linoleic acid; LOX, lipooxygenases; LT, leukotrienes; PD, protectins; PG, prostaglandins; PUFA, polyunsaturated fatty acids; Rv, resolvins; SDA, stearidonic acid; TX, thromboxanes.

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Essential fatty acids and their metabolites can exert their biological effects via multiple mechanisms. PUFA can be readily incorporated into membrane phospholipids, altering the chemical and physical properties of cell membranes and lipid rafts and thereby modulating the activity of membrane-associated functional proteins, such as ion channels and receptors [20, 21]. The eicosanoids and lipid mediators derived from PUFA can act as cell signaling messengers by binding to corresponding receptors and initiating signal transduction and gene expression; for example, prostaglandin E2 (PGE2), derived from n-6 AA, can bind to the EP2 receptor to activate pathways related to cell growth and proliferation [22]. More importantly, PUFA-derived eicosanoids and lipid mediators can serve as ligands or coactivators for a number of key transcriptional factors, such as peroxisome proliferator-activated receptors (PPARs) [23], sterol regulatory element-binding proteins [24], nuclear factor kappa B [25], and activator protein-1 [26]. Activation of these transcriptional factors has profound effects on cell proliferation and differentiation. PUFA can also influence the structure of lipid rafts in the cell membrane, and subsequently modify cellular processes such as receptor-mediated signal transduction [27-29]. Lipid rafts have been reported to play an important role in regulating stem cell self-renewal, cell cycle, survival, and induction of apoptosis [30, 31]. In addition, essential fatty acids and their metabolites are involved in energy metabolism and interact with other functional proteins or genes to affect cellular processes [32]. Overall, it is highly conceivable that PUFA and their metabolites may play a significant role in stem cell proliferation and differentiation.

Essential Fatty Acids and Stem Cell Proliferation

  1. Top of page
  2. Abstract
  3. Current Challenges in Stem Cell Therapy
  4. Essential Fatty Acid Metabolism and Cell Proliferation and Differentiation
  5. Essential Fatty Acids and Stem Cell Proliferation
  6. Essential Fatty Acids and Stem Cell Differentiation
  7. Perspectives
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References

Research on the effects of essential fatty acids on stem cell proliferation has largely focused on eicosanoids. Studies on the role of PGs, the major PUFA metabolites derived through the COX-2 pathway, in stem cell proliferation dates back to the 1970s, when E-type PGs were shown to stimulate proliferation of hematopoietic stem cells (HSCs) [33, 34]. Since then, numerous studies have supported regulatory roles for PGs and other eicosanoids in various types of stem cells, including hematopoietic, embryonic, mesenchymal, and NSCs.

PGE2, the most abundant eicosanoid derived from n-6 AA, has been shown to have profound effects on stem cell proliferation. Differential effects of PGE2 have been observed on HSCs, as it can dose-dependently inhibit growth of granulocyte/macrophage colony-forming units (CFU-GM) in vitro and myelopoiesis in vivo, but stimulate erythroid and multilineage progenitor cells [35-38] and enhance production of cycling CFU-GM from human HSCs [39]. It has been shown that treatment with PGE2 causes amplification of multipotent progenitors, enhances spleen colony-forming units, and increases the frequency of long-term repopulating HSCs in murine bone marrow after limiting dilution-competitive transplantation as well as increased kidney marrow recovery following irradiation injury in the adult zebrafish [40]. Hoggatt et al. reported that short-term ex vivo exposure of HSCs to PGE2 enhances their homing, survival, and proliferation through upregulation of the chemokine receptor CXCR4 and antiapoptotic factor survivin, in addition to enhancement of cell cycle entry and progression [41]. Indeed, treatment with a long-acting PGE2 analog after irradiation resulted in enhanced recovery of hematopoiesis and increased survival of severely irradiated mice [42, 43]. PGE2 has also been shown to directly regulate Wnt activity through cAMP/PKA-mediated regulation of β-catenin protein stability in vivo in HSCs and the hematopoietic niche during vertebrate development and organ regeneration [44]. Overall, PGE2 seems to have a proliferative effect on HSCs.

PGE2 has also been shown to affect other types of stem cells, such as ESCs and mesenchymal stem cells (MSCs). PGE2 may increase the proliferation of ESCs through upregulating the expression of cell cycle regulatory proteins and the percentage of cells in the S-phase, mediated by EP1 receptor-dependent protein kinase C (PKC) and epidermal growth factor receptor-dependent Phosphoinositide 3-kinase (PI3K)/Akt signaling pathways [45]. Mouse ESCs constitutively express COX-2 and PGE synthases to produce PGE2, which leads to resistance to apoptosis via a similar signaling pathway [46]. Interestingly, a recent study identified PGE2 as one of the small molecule compounds that, when combined, can chemically induce pluripotent stem cells from somatic cells [47]. Furthermore, PGE2 may stimulate human umbilical cord blood-derived MSC proliferation through β-catenin-mediated c-Myc and vascular endothelial growth factor expression via exchange protein directly activated by cAMP (Epac1)/Ras-related protein 1 (Rap1)/Akt and PKA cooperation [48], and through interaction of profilin-1 (Pfn-1) and filamentous-actin (F-actin) via EP2 receptor-dependent β-arrestin-1/JNK signaling pathways [22]. In addition, studies have shown that inhibitors of COX-2, an inducible enzyme for PG and TX synthesis, suppressed neurogenesis in injured brain [49-51], suggesting that PGE2 also enhances the proliferation of NSCs.

PGE1 has been shown to dose-dependently suppress colony formation of HSC with a concomitant increase in mature granulocyte production and suppression of macrophage production in vitro [52, 53]. However, PGE1 showed little or no increase in hematopoiesis in vivo [34]. Delta (12,14)-PGJ2, a natural ligand for PPARγ, dose-dependently decreases the proliferation and self-renewal in mouse ESCs by regulating leukemia inhibitory factor signaling through the JAK-STAT pathway [23]. In addition, 15d-PGJ2, an endogenous metabolite of PGD2, exhibits a biphasic regulation of epidermal growth factor-induced proliferation of NSCs derived from mouse hippocampus, that is, facilitation at low concentrations but suppression at high concentrations in vitro by modulating the redox state [54, 55].

Other classes of eicosanoids, including LT and TX, have also been investigated for their effects on stem cell proliferation. Physiological levels of LTB4 were shown to promote NSC proliferation, while excessive LTB4 levels inhibited NSC growth [56], suggesting a critical concentration range of LTB4 for NSC survival and proliferation. LTB4 was also shown to induce proliferation of HSC through interacting with BLT2 (the low affinity LTB4 receptor) and exerting antiapoptotic effects on the stem cells [57]. LTD4, which plays a key role in paracrine or autocrine regulations of embryonic and fetal functions [58], has been shown to stimulate mouse ESC proliferation and migration through the signal transducer and activator of transcription-3, PI3K/Akt, Ca2+-calcineurin, and glycogen synthase kinase 3β/β-catenin pathway [59]. Unlike LTB4 and LTD4, LXA4 and its aspirin-triggered-15-epi-LXA4 stable analog attenuated the growth of NSCs [56]. TXA2, synthesized through the n-6 COX pathway, is a vasoconstrictor and a potent hypertensive agent, and facilitates platelet aggregation. Yun et al. reported that TXA2 strongly modulated the migration and proliferation of adipose tissue-derived MSCs in a dose- and time-dependent manner through signaling mechanisms involving ERK and p38 MAPK [60].

Several studies have shown that PUFA can directly regulate stem cell proliferation. Essential fatty acids were reported to enhance early embryonic development in lactating Holstein cows, while nonessential fatty acids were inhibitory [61], suggesting a promoting effect of essential fatty acids on ESC proliferation and differentiation. Omega-6 LA was reported to enhance mouse ESC proliferation via Ca2+/PKC, PI3K/Akt, and mitogen-activated protein kinase (MAPK) signaling pathways [62]. The long-chain PUFA n-6 AA and n-3 DHA have been more thoroughly investigated for their role in NSC regulation, as they are critical for neural regeneration. Numerous studies have demonstrated that n-6 AA and n-3 DHA can enhance proliferation of NSCs [12, 63-66]. In particular, DHA has been reported to promote neurogenesis [12, 67, 68]. In short, the available evidence suggests that essential fatty acids and their metabolites, especially n-6 PUFA-derived mediators, have profound effects on the proliferation of different stem cell types.

Essential Fatty Acids and Stem Cell Differentiation

  1. Top of page
  2. Abstract
  3. Current Challenges in Stem Cell Therapy
  4. Essential Fatty Acid Metabolism and Cell Proliferation and Differentiation
  5. Essential Fatty Acids and Stem Cell Proliferation
  6. Essential Fatty Acids and Stem Cell Differentiation
  7. Perspectives
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References

Studies have demonstrated that n-3 PUFA, especially DHA, promotes NSC differentiation. DHA has been shown to enhance the differentiation of ESCs into neurons and promote neuritogenesis in vitro and in vivo, as shown by increased neurite length, neurite number, number of neurite branches, which are hallmarks of neuronal differentiation [12]. These effects were found to be associated with upregulation of the dendritic spine-related genes F-actin, GAP43, GluR1, PSD95, and synapsin-1 [12]. DHA and EPA have also been reported to induce neuronal differentiation through Hes1 and/or Hes6 pathways, which is critical for regulating NSC differentiation [68, 69]. While n-6 AA alone was found to have no effect on NSC differentiation [63], combined supplementation of DHA and AA appeared to enhance neuronal differentiation of bone marrow-derived MSCs, suggesting that DHA and AA may have a synergistic effect on NSC differentiation [70]. Furthermore, increasing the proportion of n-3 PUFA to n-6 PUFA in the diet has been shown to promote progenitor cell differentiation and reduce the frequency of myeloid progenitor cells in the bone marrow of mice [71].

In terms of the role of essential fatty acid-derived eicosanoids in stem cell differentiation, studies have mainly focused on the effects of n-6 PUFA-derived metabolites, including PGE2, LTB4, and TXA2. PGE2 has been shown to promote endothelial differentiation from bone marrow-derived cells through AMP-activated protein kinase (AMPK) activation [72], osteogenic differentiation of rat tendon stem cells via PI3K/Akt signaling [73], and differentiation of dendritic cells from CD34+ HSCs [74]. LTB4 has been shown to promote differentiation of NSCs into neurons, as indicated by enhanced neurite outgrowth upon exposure to LTB4 [56], while LT synthesis is reported to be critical for hedgehog-dependent neural differentiation of embryoid bodies [75]. TXA2 appears to be capable of inducing differentiation of human adipose tissue-derived MSCs into smooth-muscle-like cells [60, 76]. These findings indicate that the n-6 PUFA-derived eicosanoids PGE2, LTB4, and TXA2 have promoting effects on both the proliferation and differentiation of stem cells. Interestingly, one study found that inhibition of the eicosanoid synthesis pathway promotes the pluripotent state of ESCs, and that supplementation with the DHA-derived neuroprotectin D1 (NPD1), but not LTB4 or LTC4, promotes neuronal differentiation [77]. This contrast with previous findings on LTB4 may be due to differences in dosage, cell type, or culture conditions, and therefore warrants more comprehensive investigation. The finding that NPD1 supplementation enhances neuronal differentiation is consistent with the reported promoting effects of n-3 PUFA on NSC differentiation.

Perspectives

  1. Top of page
  2. Abstract
  3. Current Challenges in Stem Cell Therapy
  4. Essential Fatty Acid Metabolism and Cell Proliferation and Differentiation
  5. Essential Fatty Acids and Stem Cell Proliferation
  6. Essential Fatty Acids and Stem Cell Differentiation
  7. Perspectives
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References

It is evident that essential fatty acids can profoundly influence stem cell fate. Overall, n-6 and n-3 PUFA and their metabolites appear to exhibit promoting effects on the proliferation and differentiation of various stem cells (Table 1). Studies on n-6 PUFA have thus far focused on AA-derived eicosanoids, while research on n-3 PUFA has been limited to the effects of the fatty acids themselves (e.g., EPA and DHA). The available evidence suggests that the role of n-6 PUFA-derived metabolites in stem cell fate is mainly to enhance cell proliferation, while n-3 DHA is a promoting factor of NSC differentiation.

Given that these two classes of PUFA can generate a variety of metabolites with distinct functions, and that they act through multiple mechanisms, their overall impact is dependent on the fatty acid composition (such as the n-6/n-3 PUFA ratio) and metabolite interaction. As these factors are highly variable, it is conceivable that the specific effects of PUFA or their metabolites could differ based on cell type, metabolic state, culture conditions, and dosage. The complexity of this regulatory network is therefore both a challenge and an opportunity for stem cell research.

Understanding the role of PUFA and their metabolites in stem cell fate regulation is important for stem cell biology as well as stem cell therapy. Unlike other drugs or synthetic molecules, n-6 and n-3 PUFA are natural compounds that are safe and have numerous known health benefits. As essential nutrients obtained only through dietary intake, their tissue content in individuals can vary, but can also be easily modified through dietary intervention. Specific PUFA-derived metabolites can also be altered with drugs, such as selective COX-2 inhibitors. Therefore, modification of the fatty acid and metabolite profiles to create a favorable environment for stem cell proliferation or differentiation is a promising and practical approach to controlling stem cell fate for clinical applications. For example, PUFA supplementation could be administered prior to stem cell therapy, to either the patients or the stem cells themselves, to optimize conditions for stem cell proliferation or differentiation. Furthermore, recent studies show that administration of specific PUFA metabolites could enhance stem cell recovery from radiation injury, suggesting a potential application for PUFA metabolites as an adjunctive to radiotherapy [42, 43]. Alternatively, PUFA supplementation could be applied following stem cell delivery to facilitate proliferation or differentiation into the desired cell type.

The promise of essential fatty acids and their metabolites for stem cell fate regulation warrants further comprehensive investigation. Characterization of the specific effects of metabolites derived from n-6 and n-3 PUFA and their interactions, with regard to stem cell-related gene expression and signaling pathways, should now be performed with cutting-edge analytical technologies, such as lipidomics, metabolomics, and proteomics. Identification of potent regulating molecules that are most effective, whether alone or in combination, for specific stem cell types or conditions is also needed. Finally, development of PUFA-based intervention protocols—including formulation, dosage, and timing—is a priority for translational stem cell research.

Table 1. Effects of essential fatty acids and their metabolites on the differentiation and proliferation of stem cell models
Fatty acids/metabolitesStem cell modelsEffectsPotential pathwaysReferences
  1. Abbreviations: AA, arachidonic acid; DHA, docosahexaenoic acid; EGFR, epidermal growth factor receptor; EPA, eicosapentaenoic acid; LA, linoleic acid; LT, leukotriene; NP, neuroprotectin; PG, prostaglandin; TX, thromboxane.

LAEmbryonic stem cellsIncrease proliferationCa2+/PKC, PI3K/Akt, MAPKs[62]
AANeural stem cellsIncrease proliferationUnknown[12, 63-66]
EPANeural stem cellsPromote differentiationHes1/Hes6[69]
DHANeural stem cellsPromote differentiation, increase proliferationPI3K/Akt, RXR, PPAR, dendritic spine-related genes[12, 67-70]
PGE1Hematopoietic stem cellsInhibit proliferationUnknown[34, 52, 53]
PGE2Hematopoietic stem cellsIncrease proliferation, inhibit apoptosis, promote differentiation to dendritic cellsCXCR4, survivin, Wnt[35-44, 74]
 Embryonic stem cellsIncrease proliferation, inhibit apoptosisPI3K/Akt[45-47]
 Human umbilical cord blood-derived mesenchymal stem cellsIncrease proliferationEpac1/Rap1/Akt, α-arrestin-1/JNK[22, 48]
 Neural stem cellsIncrease proliferationUnknown[49-51]
 Bone marrow-derived cellsPromote endothelial differentiationAMPK[72]
 Tendon stem cellsPromote osteogenic differentiationPI3K/Akt[73]
Δ12,14-PGJ2Embryonic stem cellsInhibit proliferationJAK-STAT[23]
15d-PGJ2Neural stem cellsBiphasic regulation of proliferationUnknown[54, 55]
LTB4Neural stem cellsBiphasic regulation of proliferation, promote differentiation to neuronsHedgehog[56, 75]
 Hematopoietic stem cellsIncrease proliferation, inhibit apoptosisUnknown[57]
LTD4Embryonic stem cellsIncrease proliferationSTAT3, calcineurin, PI3K/Akt, GSK 3β/β-catenin[59]
LXA4Neural stem cellsInhibit proliferationEGFR, cyclin E, p27, caspase 8[56]
NPD1Embryonic stem cellsPromote neuronal differentiationUnknown[77]
TXA2Adipose tissue-derived mesenchymal stem cellsIncrease proliferation, promote differentiation to smooth-muscle-like cellsERK, p38 MAPK[60, 76]

References

  1. Top of page
  2. Abstract
  3. Current Challenges in Stem Cell Therapy
  4. Essential Fatty Acid Metabolism and Cell Proliferation and Differentiation
  5. Essential Fatty Acids and Stem Cell Proliferation
  6. Essential Fatty Acids and Stem Cell Differentiation
  7. Perspectives
  8. Acknowledgments
  9. Author Contributions
  10. Disclosure of Potential Conflicts of Interest
  11. References