Remodeled mitochondria are characteristic of pluripotent stem cells. However, a role for mitochondrial movement and distribution in pluripotency remains unknown. Here, we show that mitochondrial retrograde transport-mediated perinuclear clustering via dynein complex occurs at the early phase of pluripotency induction. Interestingly, this mitochondrial redistribution is regulated by Yamanaka factor OCT4 but not SOX2 or KLF4. This mitochondrial redistribution, which has effect on the efficiency of somatic cell reprogramming, also depends on DRP1-mediated mitochondrial fission. Importantly, perinuclear mitochondrial clustering is required for mesenchymal-to-epithelial transition (MET), an early step in reprogramming, during which β-catenin regulates the MET process. Furthermore, sufficient amount of β-catenin plays a key role in maintaining stabilization of E-CADHERIN. Taken together, these studies show that perinuclear mitochondrial clustering is an essential organellar step for MET process of pluripotency induction, which may shed light on the subcellular relationship between mitochondrial dynamics, pluripotency, and cellular morphology.

Mitochondrial diseases are historically difficult to study. They cause multi-systemic defects with prevalent impairment of hard-to-access tissues such as the brain and the heart. Furthermore, they suffer from a paucity of conventional model systems, especially because of the challenges associated with mitochondrial DNA (mtDNA) engineering. Consequently, most mitochondrial diseases are currently untreatable. Human induced pluripotent stem cells (iPSCs) represent a promising approach for developing human model systems and assessing therapeutic avenues in a patient- and tissue-specific context. iPSCs are being increasingly used to investigate mitochondrial diseases, either for dissecting mutation-specific defects within two-dimensional (2D) or three-dimensional (3D) progenies or for unveiling the impact of potential treatment options. Here, we review how iPSC-derived 2D cells and 3D organoid models have been applied to the study of mitochondrial diseases caused by either nuclear or mtDNA defects. We anticipate that the field of iPSC-driven modeling of mitochondrial diseases will continue to grow, likely leading to the development of innovative platforms for treatment discovery and toxicity that could benefit the patient community suffering from these debilitating disorders with highly unmet medical needs.

Mitochondrial dynamics, governed by fusion and fission, are crucial for maintaining cellular homeostasis, energy production, and stress adaptation. MFN2 and OPA1, key regulators of mitochondrial fusion, play essential roles beyond their structural functions, influencing bioenergetics, intracellular signaling, and quality control mechanisms such as mitophagy. Disruptions in these processes, often caused by MFN2 or OPA1 mutations, are linked to neurodegenerative diseases like Charcot-Marie-Tooth disease type 2A (CMT2A) and autosomal dominant optic atrophy (ADOA). This review explores the molecular mechanisms underlying mitochondrial fusion, the impact of MFN2 and OPA1 dysfunction on oxidative phosphorylation and autophagy, and their role in disease progression. Additionally, we discuss the divergent cellular responses to MFN2 and OPA1 mutations, particularly in terms of proliferation, senescence, and metabolic signaling. Finally, we highlight emerging therapeutic strategies to restore mitochondrial integrity, including mTOR modulation and autophagy-targeted approaches, with potential implications for neurodegenerative disorders.

Rapid developments in the field of organotypic cultures have generated a growing need for effective and non-invasive methods for quality control during tissue development. In this study, we correlate metabolic changes with epidermal differentiation and demonstrate that multiphoton microscopy with fluorescence lifetime imaging (MPM-FLIM) can be applied to monitor epidermal differentiation of keratinocytes with respect to proliferative and differentiated states. In a 2D keratinocyte tissue culture model, increased expression of differentiation markers keratin-1 and keratin-10 was induced with calcium supplementation. An accompanying shift from glycolysis to mitochondrial respiration was detected in metabolic flux assays. Analysis of MPM-FLIM images acquired at 750 nm and 900 nm excitation revealed a decreased relative fraction of intracellular NADH and FAD after high calcium treatment, consistent with increased oxidative phosphorylation. Epidermal differentiation could be monitored over a 96 h period. Discrimination analysis based on k-means clustering generated clusters that correlated well with the duration of high Ca2+ treatment, suggesting that MPM-FLIM can provide useful parameters for monitoring keratinocyte differentiation.

Autosomal recessive spinocerebellar ataxias (SCARs) are one of the most common neurodegenerative diseases characterized by progressive ataxia. Although SCARs are known to be caused by mutations in multiple genes, there are still many cases that go undiagnosed or are misdiagnosed. In this study, we presented a SCAR patient, and identified a probable novel pathogenic mutation (c.1A>G, p.M1V) in the AFG3L2 start codon. The proband's genotype included heterozygous mutations of the compound AFG3L2 (p.[M1V]; [R632X] (c.[1A>G]; [1894.C>T])), which were inherited from the father (c.1A>G, p.M1V) and mother (c.1894C>T, p.R632X). Functional studies performed on hiPSCs (human induced pluripotent stem cells) generated from the patients and HEK293T cells showed that the mutations impair mitochondrial function and the unbalanced expression of AFG3L2 mRNA and protein levels. Furthermore, this novel mutation resulted in the degradation of the protein and the reduction of the stability of the AFG3L2 protein, and MCU (mitochondrial calcium uniporter) complex mediated Ca2+ overload.

Human primary (hpBMEC) and induced pluripotent stem cell (iPSC)-derived brain microvascular endothelial-like cells (hiBMEC) are interchangeably used in blood-brain barrier models to study neurological diseases and drug delivery. Both hpBMEC and hiBMEC use glutamine as a source of carbon and nitrogen to produce metabolites and build proteins essential to cell function and communication. We used metabolomic, transcriptomic, and computational methods to examine how hpBMEC and hiBMEC metabolize glutamine, which may impact their utility in modeling the blood-brain barrier. We found that glutamine metabolism was systemically different between the two cell types. hpBMEC had a higher metabolic rate and produced more glutamate and GABA, while hiBMEC rerouted glutamine to produce more glutathione, fatty acids, and asparagine. Higher glutathione production in hiBMEC correlated with higher oxidative stress compared to hpBMEC. α-ketoglutarate (α-KG) supplementation increased glutamate secretion from hiBMEC to match that of hpBMEC; however, α-KG also decreased hiBMEC glycolytic rate. These fundamental metabolic differences between BMEC types may impact in vitro blood-brain barrier model function, particularly communication between BMEC and surrounding cells, and emphasize the importance of evaluating the metabolic impacts of iPSC-derived cells in disease models.

Neuron-glia interactions are fundamental to the development and function of the nervous system. During development, glia, including astrocytes, microglia, and oligodendrocytes, influence neuronal differentiation and migration, synapse formation and refinement, and myelination. In the mature brain, glia are crucial for maintaining neural homeostasis, modulating synaptic activity, and supporting metabolic functions. Neurons, inherently vulnerable to various stressors, rely on glia for protection and repair. However, glia, in their reactive state, can also promote neuronal damage, which contributes to neurodegenerative and neuropsychiatric diseases. Understanding the dual role of glia-as both protectors and potential aggressors-sheds light on their complex contributions to disease etiology and pathology. By appropriately modulating glial activity, it may be possible to mitigate neurodegeneration and restore neuronal function. In this review, which originated from the International Society for Neurochemistry (ISN) Advanced School in 2019 held in Montreal, Canada, we first describe the critical importance of glia in the development and maintenance of a healthy nervous system as well as their contributions to neuronal damage and neurological disorders. We then discuss potential strategies to modulate glial activity during disease to protect and promote a properly functioning nervous system. We propose that targeting glial cells presents a promising therapeutic avenue for rebuilding the nervous system.

The accumulation of damaged mitochondria has long been considered a hallmark of the aging process. Among various factors, age-related mitochondrial alterations comprise bioenergetic impairments and disturbances in reactive oxygen species (ROS) control, thereby negatively affecting mitochondrial performance and ultimately accelerating aging. Previous studies have revealed that polyamine spermidine appears to exert health-protective and lifespan-promoting effects. Notably, recent findings have also described a spermidine-induced improvement in age-associated mitochondrial dysfunction, but the beneficial effects of spermidine on aged mitochondria have not been entirely examined yet. Here, we show that spermidine positively regulates several parameters related to mitochondrial bioenergetics and mitochondrial redox homeostasis in young and aged human-induced pluripotent stem cell-derived neurons. We report that spermidine treatment increases adenosine triphosphate production and mitochondrial membrane potential, which is accompanied by an attenuation in mitochondrial ROS levels in both age groups. Furthermore, we demonstrate a spermidine-mediated amelioration in mitochondrial respiration in both young and aged neurons. Overall, our findings suggest that nutritional spermidine supplementation might represent an attractive therapeutic approach to enhance mitochondrial function, consequently decelerating aging.

Leigh syndrome (LS) is a severe mitochondrial disease that results from mutations in the nuclear or mitochondrial DNA that impairs cellular respiration and ATP production. Mutations in more than 100 genes have been demonstrated to cause LS. The disease most commonly affects brain development and function, resulting in cognitive and motor impairment. The underlying pathogenesis is challenging to ascertain due to the diverse range of symptoms exhibited by affected individuals and the variability in prognosis. To understand the disease mechanisms of different LS-causing mutations and to find a suitable treatment, several different model systems have been developed over the last 30 years. This review summarizes the established disease models of LS and their key findings. Smaller organisms such as yeast have been used to study the biochemical properties of causative mutations. Drosophila melanogaster, Danio rerio, and Caenorhabditis elegans have been used to dissect the pathophysiology of the neurological and motor symptoms of LS. Mammalian models, including the widely used Ndufs4 knockout mouse model of complex I deficiency, have been used to study the developmental, cognitive, and motor functions associated with the disease. Finally, cellular models of LS range from immortalized cell lines and trans-mitochondrial cybrids to more recent model systems such as patient-derived induced pluripotent stem cells (iPSCs). In particular, iPSCs now allow studying the effects of LS mutations in specialized human cells, including neurons, cardiomyocytes, and even three-dimensional organoids. These latter models open the possibility of developing high-throughput drug screens and personalized treatments based on defined disease characteristics captured in the context of a defined cell type. By analyzing all these different model systems, this review aims to provide an overview of past and present means to elucidate the complex pathology of LS. We conclude that each approach is valid for answering specific research questions regarding LS, and that their complementary use could be instrumental in finding treatment solutions for this severe and currently untreatable disease.

Aging represents the leading risk factor for developing neurodegenerative disorders. One of the nine hallmarks of aging is mitochondrial dysfunction. Age-related mitochondrial alterations have been shown to affect mitochondrial energy metabolism, reduction-oxidation homeostasis, and mitochondrial dynamics. Previous reports have shown that induced pluripotent stem cells (iPSCs) from aged donors do not keep the aging signature at the transcriptomic level. However, not all aspects of aging have been investigated, and especially not the mitochondria-related aging signature. Therefore, the present study compared the mitochondrial function in iPSCs from healthy aged donors compared to those of young donors. We addressed whether aged iPSCs may be used as drug-screening models of "aging in a dish" to identify therapies alleviating mitochondria aging. Compared to iPSCs from young donors, we demonstrate that iPSCs from aged donors show impaired mitochondrial bioenergetics and exhibit a rise in reactive oxygen species generation. Furthermore, aged iPSCs present a lower mitochondrial mass and alterations in the morphology of the mitochondrial network when compared to iPSCs from young donors. This study provides the first evidence that the aging phenotype is present at the mitochondrial level in iPSCs from aged donors, ranging from bioenergetics to mitochondrial network morphology. This model might be used to screen mitochondria-targeting drugs to promote healthy aging at the mitochondrial level.

Stem cells preferentially use glycolysis instead of oxidative phosphorylation and this metabolic rewiring plays an instructive role in their fate; however, the underlying molecular mechanisms remain largely unexplored. PIWI-interacting RNAs (piRNAs) and PIWI proteins have essential functions in a range of adult stem cells across species. Here, we show that piRNAs and the PIWI protein Aubergine (Aub) are instrumental in activating glycolysis in Drosophila female germline stem cells (GSCs). Higher glycolysis is required for GSC self-renewal and aub loss-of-function induces a metabolic switch in GSCs leading to their differentiation. Aub directly binds glycolytic mRNAs and Enolase mRNA regulation by Aub depends on its 5'UTR. Furthermore, mutations of a piRNA target site in Enolase 5'UTR lead to GSC loss. These data reveal an Aub/piRNA function in translational activation of glycolytic mRNAs in GSCs, and pinpoint a mechanism of regulation of metabolic reprogramming in stem cells based on small RNAs.

Frequent recurrence and metastasis caused by cancer stem cells (CSCs) are major challenges in lung cancer treatment. Therefore, identifying and characterizing specific CSC targets are crucial for the success of prospective targeted therapies. In this study, it is found that upregulated TOR Signaling Pathway Regulator-Like (TIPRL) in lung CSCs causes sustained activation of the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) signaling pathway by binding to CaMKK2, thereby maintaining stemness and survival. CaMKK2-mediated activation of CaM kinase 4 (CaMK4) leads to phosphorylation of cAMP response element-binding protein (CREB) at Ser129 and Ser133, which is necessary for its maximum activation and the downstream constitutive expression of its target genes (Bcl2 and HMG20A). TIPRL depletion sensitizes lung CSCs to afatinib-induced cell death and reduces distal metastasis of lung cancer in vivo. It is determined that CREB activates the transcription of TIPRL in lung CSCs. The positive feedback loop consisting of CREB and TIPRL induces the sustained activation of the CaMKK2-CaMK4-CREB axis as a driving force and upregulates the expression of stemness- and survival-related genes, promoting tumorigenesis in patients with lung cancer. Thus, TIPRL and the CaMKK2 signaling axis may be promising targets for overcoming drug resistance and reducing metastasis in lung cancer.

With the use of specific genetic factors and recent developments in cellular reprogramming, it is now possible to generate lineage-committed cells or induced pluripotent stem cells (iPSCs) from readily available and common somatic cell types. However, there are still significant doubts regarding the safety and effectiveness of the current genetic methods for reprogramming cells, as well as the conventional culture methods for maintaining stem cells. Small molecules that target specific epigenetic processes, signaling pathways, and other cellular processes can be used as a complementary approach to manipulate cell fate to achieve a desired objective. It has been discovered that a growing number of small molecules can support lineage differentiation, maintain stem cell self-renewal potential, and facilitate reprogramming by either increasing the efficiency of reprogramming or acting as a genetic reprogramming factor substitute. However, ongoing challenges include improving reprogramming efficiency, ensuring the safety of small molecules, and addressing issues with incomplete epigenetic resetting. Small molecule iPSCs have significant clinical applications in regenerative medicine and personalized therapies. This review emphasizes the versatility and potential safety benefits of small molecules in overcoming challenges associated with the iPSCs reprogramming process.

Cancer stem cells (CSCs) are a subset of tumor cells that can initiate and sustain tumor growth and cause recurrence and metastasis. CSCs are particularly resistant to conventional therapies compared to their counterparts, owing greatly to their intrinsic metabolic plasticity. Metabolic plasticity allows CSCs to switch between different energy production and usage pathways based on environmental and extrinsic factors, including conditions imposed by conventional cancer therapies. To cope with nutrient deprivation and therapeutic stress, CSCs can transpose between glycolysis and oxidative phosphorylation (OXPHOS) metabolism. The mechanism behind the metabolic pathway switch in CSCs is not fully understood, however, some evidence suggests that the tumor microenvironment (TME) may play an influential role mediated by its release of signals, such as Wnt/β-catenin and Notch pathways, as well as a background of hypoxia. Exploring the factors that promote metabolic plasticity in CSCs offers the possibility of eventually developing therapies that may more effectively eliminate the crucial tumor cell subtype and alter the disease course substantially.

With the elderly population projected to double by 2050, there is an urgent need to address the increasing prevalence of age-related debilitating diseases and ultimately minimize discrepancies between the rising lifespan and stagnant health span. Cellular reprogramming by overexpression of Oct3/4, Klf4, Sox2, and cMyc (OKSM) transcription factors is gaining attention in this context thanks to demonstrated rejuvenating effects in human cell cultures and live mice, many of which can be uncoupled from dedifferentiation and loss of cell identity.

Here, we review current evidence of the impact of cell reprogramming on established aging hallmarks and the underlying mechanisms that mediate these effects. We also provide a critical assessment of the challenges in translating these findings and, overall, cell reprogramming technologies into clinically translatable antiaging interventions.

Cellular reprogramming has the potential to reverse at least partially some key hallmarks of aging. However, further research is necessary to determine the biological significance and duration of such changes and to ensure the safety of cell reprogramming as a rejuvenation approach. With this review, we hope to stimulate new research directions in the quest to extend health span effectively.

Embryonic stem cells (ESCs) exhibit a metabolic preference for glycolysis over oxidative phosphorylation to meet their substantial adenosine triphosphate (ATP) demands during self-renewal. This metabolic choice inherently maintains low mitochondrial activity and minimal reactive oxygen species (ROS) generation. Nonetheless, the intricate molecular mechanisms governing the restraint of ROS production and the mitigation of cellular damage remain incompletely elucidated. In this study, we reveal the pivotal role of RNA-binding motif protein 46 (RBM46) in ESCs, acting as a direct post transcriptional regulator of ROS levels by modulating BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) mRNA expression. Rbm46 knockout lead to diminished mitochondrial autophagy, culminating in elevated ROS within ESCs, disrupting the delicate balance required for healthy self-renewal. These findings provide insights into a novel mechanism governing ROS regulation in ESCs.

Mitochondria are the energy factories of a cell, and depending on the metabolic requirements, the mitochondrial morphology, quantity, and membrane potential in a cell change. These changes are frequently assessed using commercially available probes. In this study, we tested the suitability of three commercially available probes-namely 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolo-carbocyanine iodide (JC-1), MitoTracker Red CMX Rox (CMXRos), and tetramethylrhodamine methyl ester (TMRM)-for assessing the mitochondrial quantity, morphology, and membrane potential in living human mesoangioblasts in 3D with confocal laser scanning microscope (CLSM) and scanning disk confocal microscope (SDCM). Using CLSM, JC-1, and CMXRos-but not TMRM-uncovered considerable background and variation. Using SDCM, the background signal only remained apparent for the JC-1 monomer. Repetitive imaging of CMXRos and JC-1-but not TMRM-demonstrated a 1.5-2-fold variation in signal intensity between cells using CLSM. The use of SDCM drastically reduced this variation. The slope of the relative signal intensity upon repetitive imaging using CLSM was lowest for TMRM (-0.03) and highest for CMXRos (0.16). Upon repetitive imaging using SDCM, the slope varied from 0 (CMXRos) to a maximum of -0.27 (JC-1 C1). Conclusively, our data show that TMRM staining outperformed JC-1 and CMXRos dyes in a (repetitive) 3D analysis of the entire mitochondrial quantity, morphology, and membrane potential in living cells.

Enteroendocrine cells (EECs) are crucial for sensing ingested nutrients and regulating feeding behavior. How gut microbiota regulate the nutrient-sensing EEC activity is unclear. Our transcriptomic analysis demonstrates that commensal microbiota colonization significantly increases the expression of many genes associated with mitochondrial function. Using new methods to image EEC cytoplasmic and mitochondrial Ca2+ activity in live zebrafish, our data revealed that it is dynamically regulated during the EEC development process. Mature EECs display an increased mitochondrial-to-cytoplasmic Ca2+ ratio. Mitochondria are evenly distributed in the cytoplasm of immature EECs. As EECs mature, their mitochondria are highly localized at the basal membrane where EEC vesicle secretion occurs. Conventionalized (CV) EECs, but not germ-free (GF) EECs, exhibit spontaneous low-amplitude Ca2+ fluctuation. The mitochondrial-to-cytoplasmic Ca2+ ratio is significantly higher in CV EECs. Nutrient stimulants, such as fatty acid, increase cytoplasmic Ca2+ in a subset of EECs and promote a sustained mitochondrial Ca2+ and ATP increase. However, the nutrient-induced EEC mitochondrial activation is nearly abolished in GF zebrafish. Together, our study reveals that commensal microbiota are crucial in supporting EEC mitochondrial function and maturation.

Pluripotent stem cells of the mammalian epiblast and their cultured counterparts-embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs)-have the capacity to differentiate in all cell types of adult organisms. An artificial process of reactivation of the pluripotency program in terminally differentiated cells was established in 2006, which allowed for the generation of induced pluripotent stem cells (iPSCs). This iPSC technology has become an invaluable tool in investigating the molecular mechanisms of human diseases and therapeutic drug development, and it also holds tremendous promise for iPSC applications in regenerative medicine. Since the process of induced reprogramming of differentiated cells to a pluripotent state was discovered, many questions about the molecular mechanisms involved in this process have been clarified. Studies conducted over the past 2 decades have established that metabolic pathways and retrograde mitochondrial signals are involved in the regulation of various aspects of stem cell biology, including differentiation, pluripotency acquisition, and maintenance. During the reprogramming process, cells undergo major transformations, progressing through three distinct stages that are regulated by different signaling pathways, transcription factor networks, and inputs from metabolic pathways. Among the main metabolic features of this process, representing a switch from the dominance of oxidative phosphorylation to aerobic glycolysis and anabolic processes, are many critical stage-specific metabolic signals that control the path of differentiated cells toward a pluripotent state. In this review, we discuss the achievements in the current understanding of the molecular mechanisms of processes controlled by metabolic pathways, and vice versa, during the reprogramming process.

Mitochondria, classically known as the powerhouse of cells, are unique double membrane-bound multifaceted organelles carrying a genome. Mitochondrial content varies between cell types and precisely doubles within cells during each proliferating cycle. Mitochondrial content also increases to a variable degree during cell differentiation triggered after exit from the proliferating cycle. The mitochondrial content is primarily maintained by the regulation of mitochondrial biogenesis, while damaged mitochondria are eliminated from the cells by mitophagy. In any cell with a given mitochondrial content, the steady-state mitochondrial number and shape are determined by a balance between mitochondrial fission and fusion processes. The increase in mitochondrial content and alteration in mitochondrial fission and fusion are causatively linked with the process of differentiation. Here, we critically review the quantitative aspects in the detection methods of mitochondrial content and shape. Thereafter, we quantitatively link these mitochondrial properties in differentiating cells and highlight the implications of such quantitative link on stem cell functionality. Finally, we discuss an example of cell size regulation predicted from quantitative analysis of mitochondrial shape and content. To highlight the significance of quantitative analyses of these mitochondrial properties, we propose three independent rationale based hypotheses and the relevant experimental designs to test them.

Metabolic programs in the stem cells are essential for maintaining homeostasis and protecting against stem cell aging. There is growing evidence that the tissue stem cells reside in the anterior and posterior maculae flavae of the human vocal fold mucosa. Our previous studies observed that the glycolysis of the cell in the human maculae flavae seems to rely more on anaerobic glycolysis for energy supply in comparison with oxidative phosphorylation. However, previous studies showed only the metabolic enzymes of glycolysis and functional morphology of the mitochondria, therefore, it has not yet been determined whether anaerobic glycolysis actually took place. The purpose of this study is to investigate the glycolytic metabolites of the cells in the maculae flavae of the human vocal fold in vitro.

Four normal human vocal folds were used. After extraction of the anterior maculae flavae, cells in the maculae flavae were cultured and proliferated. Glucose transporter-1 was assessed using immunocytochemistry and metabolites of glycolysis (lactate and NADPH) were measured.

The cells in the maculae flavae expressed glucose transporter-1 in the cytoplasm and the cell membranes. In addition, the cultured cells produced lactate (metabolites of anaerobic glycolysis) and NADPH (metabolites of the pentose phosphate pathway).

The cells in the maculae flavae of the human vocal folds were found to undergo anaerobic glycolysis via the pentose phosphate pathway. This suggests that the cells in the maculae flavae of the human vocal fold have a metabolism that favors the maintenance of stemness and undifferentiated states.

The enteroendocrine cells (EECs) in the intestine are crucial for sensing ingested nutrients and regulating feeding behavior. The means by which gut microbiota regulates the nutrient-sensing EEC activity is unclear. Our transcriptomic analysis of the EECs from germ-free (GF) and conventionalized (CV) zebrafish revealed that commensal microbiota colonization significantly increased the expression of many genes that are associated with mitochondrial function. Using in vivo imaging and 3D automated cell tracking approach, we developed new methods to image and analyze the EECs' cytoplasmic and mitochondrial calcium activity at cellular resolution in live zebrafish. Our data revealed that during the development, shortly after gut microbiota colonization, EECs briefly increased cytoplasm and mitochondrial Ca2+, a phenomenon we referred to as "EEC awakening". Following the EEC awakening, cytoplasmic Ca2+ levels but not mitochondrial Ca2+ level in the EECs decreased, resulting in a consistent increase in the mitochondrial-to-cytoplasmic Ca2+ ratio. The increased mitochondrial-to-cytoplasmic Ca2+ ratio is associated with the EEC maturation process. In immature EECs, we further discovered that their mitochondria are evenly distributed in the cytoplasm. When EECs mature, their mitochondria are highly localized in the basal lateral membrane where EEC vesicle secretion occurs. Furthermore, CV EECs, but not GF EECs, exhibit spontaneous low-amplitude calcium fluctuation. The mitochondrial-to-cytoplasm Ca2+ ratio is significantly higher in CV EECs. When stimulating the CV zebrafish with nutrients like fatty acids, nutrient stimulants increase cytoplasmic Ca2+ in a subset of EECs and promote a sustained mitochondrial Ca2+ increase. However, the nutrient induced EEC mitochondrial activation is nearly abolished in GF zebrafish. Together, our study reveals that commensal microbiota are critical in supporting EEC mitochondrial function and maturation. Selectively manipulating gut microbial signals to alter EEC mitochondrial function will provide new opportunities to change gut-brain nutrient sensing efficiency and feeding behavior.

Mitochondria are the powerhouse of the cell and dynamically control fundamental biological processes including cell reprogramming, pluripotency, and lineage specification. Although remarkable progress in induced pluripotent stem cell (iPSC)-derived cell therapies has been made, very little is known about the role of mitochondria and the mechanisms involved in somatic cell reprogramming into iPSC and directed reprogramming of iPSCs in terminally differentiated cells. Reprogramming requires changes in cellular characteristics, genomic and epigenetic regulation, as well as major mitochondrial metabolic changes to sustain iPSC self-renewal, pluripotency, and proliferation. Differentiation of autologous iPSC into terminally differentiated β-like cells requires further metabolic adaptation. Many studies have characterized these alterations in signaling pathways required for the generation and differentiation of iPSC; however, very little is known regarding the metabolic shifts that govern pluripotency transition to tissue-specific lineage differentiation. Understanding such metabolic transitions and how to modulate them is essential for the optimization of differentiation processes to ensure safe iPSC-derived cell therapies. In this review, we summarize the current understanding of mitochondrial metabolism during somatic cell reprogramming to iPSCs and the metabolic shift that occurs during directed differentiation into pancreatic β-like cells.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are ER Ca2+-release channels that control a broad set of cellular processes. Animal models lacking IP3Rs in different combinations display severe developmental phenotypes. Given the importance of IP3Rs in human diseases, we investigated their role in human induced pluripotent stem cells (hiPSC) by developing single IP3R and triple IP3R knockouts (TKO). Genome edited TKO-hiPSC lacking all three IP3R isoforms, IP3R1, IP3R2, IP3R3, failed to generate Ca2+ signals in response to agonists activating GPCRs, but retained stemness and pluripotency. Steady state metabolite profiling and flux analysis of TKO-hiPSC indicated distinct alterations in tricarboxylic acid cycle metabolites consistent with a deficiency in their pyruvate utilization via pyruvate dehydrogenase, shifting towards pyruvate carboxylase pathway. These results demonstrate that IP3Rs are not essential for hiPSC identity and pluripotency but regulate mitochondrial metabolism. This set of knockout hiPSC is a valuable resource for investigating IP3Rs in human cell types of interest.

The term 'cloning' refers to the production of genetically identical individuals but has meant different things throughout the history of science: a natural means of reproduction in bacteria, a routine procedure in horticulture, and an ever-evolving gamut of molecular technologies in vertebrates. Mammalian cloning can be achieved through embryo splitting, somatic cell nuclear transfer, and most recently, by the use of induced pluripotent stem cells. Several emerging biotechnologies also facilitate the propagation of genomes from one generation to the next whilst bypassing the conventional reproductive processes. In this review, we examine the state of the art of available cloning technologies and their progress in species other than humans and rodent models, in order to provide a critical overview of their readiness and relevance for application in endangered animal conservation.

Antibiotics inhibit breast cancer stem cells (CSCs) by suppressing mitochondrial biogenesis. However, the effectiveness of antibiotics in clinical settings is inconsistent. This inconsistency raises the question of whether the tumor microenvironment, particularly hypoxia, plays a role in the response to antibiotics. Therefore, the goal of this study was to evaluate the effectiveness of five commonly used antibiotics for inhibiting CSCs under hypoxia using an MCF-7 cell line model. We assessed the number of CSCs through the mammosphere formation assay and aldehyde dehydrogenase (ALDH)-bright cell count. Additionally, we examined the impact of antibiotics on the mitochondrial stress response and membrane potential. Furthermore, we analyzed the levels of proteins associated with therapeutic resistance. There was no significant difference in the number of CSCs between cells cultured under normoxic and hypoxic conditions. However, hypoxia did affect the rate of CSC inhibition by antibiotics. Specifically, azithromycin was unable to inhibit sphere formation in hypoxia. Erythromycin and doxycycline did not reduce the ratio of ALDH-bright cells, despite decreasing the number of mammospheres. Furthermore, treatment with chloramphenicol, doxycycline, and tetracycline led to the overexpression of the breast cancer resistance protein. Our findings suggest that hypoxia may weaken the inhibitory effects of antibiotics on the breast cancer model.

Most age-related neurodegenerative diseases remain incurable owing to an incomplete understanding of the disease mechanisms. Several environmental and genetic factors contribute to disease onset, with human biological ageing being the primary risk factor. In response to acute cellular damage and external stimuli, somatic cells undergo state shifts characterized by temporal changes in their structure and function that increase their resilience, repair cellular damage, and lead to their mobilization to counteract the pathology. This basic cell biological principle also applies to human brain cells, including mature neurons that upregulate developmental features such as cell cycle markers or glycolytic reprogramming in response to stress. Although such temporary state shifts are required to sustain the function and resilience of the young human brain, excessive state shifts in the aged brain might result in terminal fate loss of neurons and glia, characterized by a permanent change in cell identity. Here, we offer a new perspective on the roles of cell states in sustaining health and counteracting disease, and we examine how cellular ageing might set the stage for pathological fate loss and neurodegeneration. A better understanding of neuronal state and fate shifts might provide the means for a controlled manipulation of cell fate to promote brain resilience and repair.

Fibroblasts can be chemically induced to pluripotent stem cells (CiPSCs) through an extraembryonic endoderm (XEN)-like state or directly converted into other differentiated cell lineages. However, the mechanisms underlying chemically induced cell-fate reprogramming remain unclear. Here, a transcriptome-based screen of biologically active compounds uncovered that CDK8 inhibition was essential to enable chemically induced reprogramming from fibroblasts into XEN-like cells, then CiPSCs. RNA-sequencing analysis showed that CDK8 inhibition downregulated proinflammatory pathways that suppress chemical reprogramming and facilitated the induction of a multi-lineage priming state, indicating the establishment of plasticity in fibroblasts. CDK8 inhibition also resulted in a chromatin accessibility profile like that under initial chemical reprogramming. Moreover, CDK8 inhibition greatly promoted reprogramming of mouse fibroblasts into hepatocyte-like cells and induction of human fibroblasts into adipocytes. These collective findings thus highlight CDK8 as a general molecular barrier in multiple cell reprogramming processes, and as a common target for inducing plasticity and cell fate conversion.

Homozygous or compound heterozygous (biallelic) variants in PRKN are causal for PD with highly penetrant symptom expression, while the much more common heterozygous variants may predispose to PD with highly reduced penetrance, through altered mitochondrial function. In the presence of pathogenic heterozygous variants, it is therefore important to test for mitochondrial alteration in cells derived from variant carriers to establish potential presymptomatic molecular markers. We generated lymphoblasts (LCLs) and human induced pluripotent stem cell (hiPSC)-derived neurons from non-manifesting heterozygous PRKN variant carriers and tested them for mitochondrial functionality. In LCLs, we detected hyperactive mitochondrial respiration, and, although milder compared to a biallelic PRKN-PD patient, hiPSC-derived neurons of non-manifesting heterozygous variant carriers also displayed several phenotypes of altered mitochondrial function. Overall, we identified molecular phenotypes that might be used to monitor heterozygous PRKN variant carriers during the prodromal phase. Such markers might also be useful to identify individuals at greater risk of eventual disease development and for testing potential mitochondrial function-based neuroprotective therapies before neurodegeneration advances.

Post-translational modifications of proteins are crucial to the regulation of their activity and function. As a newly discovered acylation modification, crotonylation of non-histone proteins remains largely unexplored, particularly in human embryonic stem cells (hESCs).

We investigated the role of crotonylation in hESC differentiation by introduce crotonate into the culture medium of GFP tagged LTR7 primed H9 cell and extended pluripotent stem cell lines. RNA-seq assay was used to determine the hESC transcriptional features. Through morphological changes, qPCR of pluripotent and germ layer-specific gene markers and flow cytometry analysis, we determined that the induced crotonylation resulted in hESC differentiating into the endodermal lineage. We performed targeted metabolomic analysis and seahorse metabolic measurement to investigate the metabolism features after crotonate induction. Then high-resolution tandem mass spectrometry (LC-MS/MS) revealed the target proteins in hESCs. In addition, the role of crotonylated glycolytic enzymes (GAPDH and ENOA) was evaluated by in vitro crotonylation and enzymatic activity assays. Finally, we used knocked-down hESCs by shRNA, wild GAPDH and GAPDH mutants to explore potential role of GAPDH crotonylation in regulating human embryonic stem cell differentiation and metabolic switch.

We found that induced crotonylation in hESCs resulted in hESCs of different pluripotency states differentiating into the endodermal lineage. Increased protein crotonylation in hESCs was accompanied by transcriptomic shifts and decreased glycolysis. Large-scale crotonylation profiling of non-histone proteins revealed that metabolic enzymes were major targets of inducible crotonylation in hESCs. We further discovered GAPDH as a key glycolytic enzyme regulated by crotonylation during endodermal differentiation from hESCs.

Crotonylation of GAPDH decreased its enzymatic activity thereby leading to reduced glycolysis during endodermal differentiation from hESCs.

The ageing process is a systemic decline from cellular dysfunction to organ degeneration, with more predisposition to deteriorated disorders. Rejuvenation refers to giving aged cells or organisms more youthful characteristics through various techniques, such as cellular reprogramming and epigenetic regulation. The great leaps in cellular rejuvenation prove that ageing is not a one-way street, and many rejuvenative interventions have emerged to delay and even reverse the ageing process. Defining the mechanism by which roadblocks and signaling inputs influence complex ageing programs is essential for understanding and developing rejuvenative strategies. Here, we discuss the intrinsic and extrinsic factors that counteract cell rejuvenation, and the targeted cells and core mechanisms involved in this process. Then, we critically summarize the latest advances in state-of-art strategies of cellular rejuvenation. Various rejuvenation methods also provide insights for treating specific ageing-related diseases, including cellular reprogramming, the removal of senescence cells (SCs) and suppression of senescence-associated secretory phenotype (SASP), metabolic manipulation, stem cells-associated therapy, dietary restriction, immune rejuvenation and heterochronic transplantation, etc. The potential applications of rejuvenation therapy also extend to cancer treatment. Finally, we analyze in detail the therapeutic opportunities and challenges of rejuvenation technology. Deciphering rejuvenation interventions will provide further insights into anti-ageing and ageing-related disease treatment in clinical settings.

Mitochondrial DNA (mtDNA) diseases are multi-systemic disorders caused by mutations affecting a fraction or the entirety of mtDNA copies. Currently, there are no approved therapies for the majority of mtDNA diseases. Challenges associated with engineering mtDNA have in fact hindered the study of mtDNA defects. Despite these difficulties, it has been possible to develop valuable cellular and animal models of mtDNA diseases. Here, we describe recent advances in base editing of mtDNA and the generation of three-dimensional organoids from patient-derived human-induced pluripotent stem cells (iPSCs). Together with already available modeling tools, the combination of these novel technologies could allow determining the impact of specific mtDNA mutations in distinct human cell types and might help uncover how mtDNA mutation load segregates during tissue organization. iPSC-derived organoids could also represent a platform for the identification of treatment strategies and for probing the in vitro effectiveness of mtDNA gene therapies. These studies have the potential to increase our mechanistic understanding of mtDNA diseases and may open the way to highly needed and personalized therapeutic interventions.

Cellular redox state directs differentiation of induced pluripotent stem cells (iPSC) by energy metabolism control and ROS generation. As oxidative stress and mitochondrial dysfunction have been extensively reported in Down syndrome (DS), we evaluated mitochondrial phenotypes and energy metabolism during neural differentiation of DS iPSCs to neural progenitor cells (NPCs). Our results indicate early maturation of mitochondrial networks and elevated NADPH oxidase 4 (NOX4) expression in DS iPSCs. DS cells also fail to transition from glycolysis to oxidative phosphorylation during differentiation. Specifically, DS NPCs show an increased energetic demand that is limited in their mitochondrial and glycolytic response to mitochondrial distress. Additionally, DS iPSC and NPC non-mitochondrial oxygen consumption was significantly impacted by NOX inhibition. Together, these data build upon previous evidence of accelerated neural differentiation in DS that correlates with cellular redox state. We demonstrate the potential for mitochondrial and non-mitochondrial ROS sources to impact differentiation timing in the context of DS, which could contribute to developmental deficits in this condition.

Since the discovery of Neural Stem Cells (NSCs) there are still mechanism to be clarified, such as the role of mitochondrial metabolism in the regulation of endogenous adult neurogenesis and its implication in neurodegeneration. Although stem cells require glycolysis to maintain their stemness, they can perform oxidative phosphorylation and it is becoming more and more evident that mitochondria are central players, not only for ATP production but also for neuronal differentiation's steps regulation, through their ability to handle cellular redox state, intracellular signaling, epigenetic state of the cell, as well as the gut microbiota-brain axis, upon dietary influences. In this scenario, the 8-oxoguanine DNA glycosylase (OGG1) repair system would link mitochondrial DNA integrity to the modulation of neural differentiation. On the other side, there is an increasing interest in NSCs generation, from induced pluripotent stem cells, as a clinical model for neurodegenerative diseases (NDs), although this methodology still presents several drawbacks, mainly related to the reprogramming process. Indeed, high levels of reactive oxygen species (ROS), associated with telomere shortening, genomic instability, and defective mitochondrial dynamics, lead to pluripotency limitation and reprogramming efficiency's reduction. Moreover, while a physiological or moderate ROS increase serves as a signaling mechanism, to activate differentiation and suppress self-renewal, excessive oxidative stress is a common feature of NDs and aging. This ROS-dependent regulatory effect might be modulated by newly identified ROS suppressors, including the NAD⁺-dependent deacetylase enzymes family called Sirtuins (SIRTs). Recently, the importance of subcellular localization of NAD synthesis has been coupled to different roles for NAD in chromatin stability, DNA repair, circadian rhythms, and longevity. SIRTs have been described as involved in the control of both telomere's chromatin state and expression of nuclear gene involved in the regulation of mitochondrial gene expression, as well as in several NDs and aging. SIRTs are ubiquitously expressed in the mammalian brain, where they play important roles. In this review we summarize the current knowledge on how SIRTs-dependent modulation of mitochondrial metabolism could impact on neurogenesis and neurodegeneration, focusing mainly on ROS function and their role in SIRTs-mediated cell reprogramming and telomere protection.

The formation of embryoid bodies (EBs) from human pluripotent stem cells resembles the early stages of human embryo development, mimicking the organization of three germ layers. In our study, EBs were tested for their vulnerability to chronic exposure to low doses of MeHgCl (1 nM) under atmospheric (21%O₂) and physioxia (5%O₂) conditions. Significant differences were observed in the relative expression of genes associated with DNA repair and mitophagy between the tested oxygen conditions in nontreated EBs. When compared to physioxia conditions, the significant differences recorded in EBs cultured at 21% O₂ included: (1) lower expression of genes associated with DNA repair (ATM, OGG1, PARP1, POLG1) and mitophagy (PARK2); (2) higher level of mtDNA copy number; and (3) higher expression of the neuroectodermal gene (NES). Chronic exposure to a low dose of MeHgCl (1 nM) disrupted the development of EBs under both oxygen conditions. However, only EBs exposed to MeHgCl at 21% O₂ revealed downregulation of mtDNA copy number, increased oxidative DNA damage and DNA fragmentation, as well as disturbances in SOX17 (endoderm) and TBXT (mesoderm) genes expression. Our data revealed that physioxia conditions protected EBs genome integrity and their further differentiation.

The vascularization of engineered tissues and organoids has remained a major unresolved challenge in regenerative medicine. While multiple approaches have been developed to vascularize in vitro tissues, it has thus far not been possible to generate sufficiently dense networks of small-scale vessels to perfuse large de novo tissues. Here, we achieve the perfusion of multi-mm3 tissue constructs by generating networks of synthetic capillary-scale 3D vessels. Our 3D soft microfluidic strategy is uniquely enabled by a 3D-printable 2-photon-polymerizable hydrogel formulation, which allows for precise microvessel printing at scales below the diffusion limit of living tissues. We demonstrate that these large-scale engineered tissues are viable, proliferative and exhibit complex morphogenesis during long-term in-vitro culture, while avoiding hypoxia and necrosis. We show by scRNAseq and immunohistochemistry that neural differentiation is significantly accelerated in perfused neural constructs. Additionally, we illustrate the versatility of this platform by demonstrating long-term perfusion of developing neural and liver tissue. This fully synthetic vascularization platform opens the door to the generation of human tissue models at unprecedented scale and complexity.

Metabolism shifts from glycolysis to mitochondrial oxidative phosphorylation are vital during the differentiation of stem cells. Mitochondria have a direct function in differentiation. However, the metabolic shift and the effect of mitochondria in regulating the osteogenic differentiation of human dental pulp stem cells (hDPSCs) remain unclear.

Human dental pulp stem cells were collected from five healthy donors. Osteogenic differentiation was induced by osteogenic induction medium. The activities of alkaline phosphatase, hexokinase, pyruvate kinase, and lactate dehydrogenase were analyzed by enzymatic activity kits. The extracellular acidification rate and the mitochondrial oxygen consumption rate were measured. The mRNA levels of COL-1, ALP, TFAM, and NRF1 were analyzed. The protein levels of p-AMPK and AMPK were detected by western blotting.

Glycolysis decreased after a slight increase, while mitochondrial oxidative phosphorylation continued to increase when cells grew in osteogenic induction medium. Therefore, the metabolism of differentiating cells switched to mitochondrial respiration. Next, inhibiting mitochondrial respiration with carbonyl cyanide-chlorophenylhydrazone, a mitochondrial uncoupler inhibited hDPSCs differentiation with less ALP activity and decreased ALP and COL-1 mRNA expression. Furthermore, mitochondrial uncoupling led to AMPK activation. 5-Aminoimidazole-4-carboxamide ribonucleotide, an AMPK activator, simulated the effect of mitochondrial uncoupling by inhibiting osteogenic differentiation, mitochondrial biogenesis, and mitochondrial morphology. Mitochondrial uncoupling and activation of AMPK depressed mitochondrial oxidative phosphorylation and inhibited differentiation, suggesting that they may serve as regulators to halt osteogenic differentiation from impaired mitochondrial oxidative phosphorylation.

Mitochondria are indispensable power plants of eukaryotic cells that also act as a major biochemical hub. As such, mitochondrial dysfunction, which can originate from mutations in the mitochondrial genome (mtDNA), may impair organism fitness and lead to severe diseases in humans. MtDNA is a multi-copy, highly polymorphic genome that is uniparentally transmitted through the maternal line. Several mechanisms act in the germline to counteract heteroplasmy (i.e., coexistence of two or more mtDNA variants) and prevent expansion of mtDNA mutations. However, reproductive biotechnologies such as cloning by nuclear transfer can disrupt mtDNA inheritance, resulting in new genetic combinations that may be unstable and have physiological consequences. Here, we review the current understanding of mitochondrial inheritance, with emphasis on its pattern in animals and human embryos generated by nuclear transfer.

Stem cells are defined by their ability to self-renew and differentiate, both shown in multiple studies to be regulated by metabolic processes. To decipher metabolic signatures of self-renewal in blastocyst-derived stem cells, we compared early differentiating embryonic stem cells (ESCs) and their extra-embryonic counterparts, trophoblast (T)SCs to their self-renewing counterparts. A metabolomics analysis pointed to the desaturation of fatty acyl chains as a metabolic signature of differentiating blastocyst-derived SCs via the upregulation of delta-6 desaturase (D6D; FADS2) and delta-5 desaturase (D5D; FADS1), key enzymes in the biosynthesis of polyunsaturated fatty acids (PUFAs). The inhibition of D6D or D5D by specific inhibitors or SiRNA retained stemness in ESCs and TSCs, and attenuated endoplasmic reticulum (ER) stress-related apoptosis. D6D inhibition in ESCs upregulated stearoyl-CoA desaturase-1 (Scd1), essential to maintain ER homeostasis. In TSCs, however, D6D inhibition downregulated Scd1. TSCs show higher Scd1 mRNA expression and high levels of monounsaturated fatty acyl chain products in comparison to ESCs. The addition of oleic acid, the product of Scd1 (essential for ESCs), to culture medium, was detrimental to TSCs. Interestingly, TSCs express a high molecular mass variant of Scd1 protein, hardly expressed by ESCs. Taken together, our data suggest that lipid desaturation is a metabolic regulator of the balance between differentiation and self-renewal of ESCs and TSCs. They point to lipid polydesaturation as a driver of differentiation in both cell types. Monounsaturated fatty acids (MUFAs), essential for ESCs are detrimental to TSCs.

While the U.S. Food and Drug Administration and the European Medicines Evaluation Agency have recently approved new drugs to treat spinal muscular atrophy 1 (SMA1) in young patients, they are mostly ineffective in older patients since many motor neurons have already been lost. Therefore, understanding nervous system (NS) physiology in SMA patients is essential. Consequently, studying neural stem cells (NSCs) from SMA patients is of significant interest in searching for new treatment targets that will enable researchers to identify new pharmacological approaches. However, studying NSCs in these patients is challenging since their isolation damages the NS, making it impossible with living patients. Nevertheless, it is possible to study NSCs from animal models or create them by differentiating induced pluripotent stem cells obtained from SMA patient peripheral tissues. On the other hand, therapeutic interventions such as NSCs transplantation could ameliorate SMA condition. This review summarizes current knowledge on the physiological properties of NSCs from animals and human cellular models with an SMA background converging on the molecular and neuronal circuit formation alterations of SMA fetuses and is not focused on the treatment of SMA. By understanding how SMA alters NSC physiology, we can identify new and promising interventions that could help support affected patients.

Embryonic stem cells (ESCs) have a significantly lower mutation load compared to somatic cells, but the mechanisms that guard genomic integrity in ESCs remain largely unknown. Here we show that BNIP3-dependent mitophagy protects genomic integrity in mouse ESCs. Deletion of Bnip3 increases cellular reactive oxygen species (ROS) and decreases ATP generation. Increased ROS in Bnip3^-/- ESCs compromised self-renewal and were partially rescued by either NAC treatment or p53 depletion. The decreased cellular ATP in Bnip3^-/- ESCs induced AMPK activation and deteriorated homologous recombination, leading to elevated mutation load during long-term propagation. Whereas activation of AMPK in X-ray-treated Bnip3^+/+ ESCs dramatically ascended mutation rates, inactivation of AMPK in Bnip3^-/- ESCs under X-ray stress remarkably decreased the mutation load. In addition, enhancement of BNIP3-dependent mitophagy during reprogramming markedly decreased mutation accumulation in established iPSCs. In conclusion, we demonstrated a novel pathway in which BNIP3-dependent mitophagy safeguards ESC genomic stability, and that could potentially be targeted to improve pluripotent stem cell genomic integrity for regenerative medicine.