Mitochondrial regulation in human pluripotent stem cells during reprogramming and β cell differentiation

Abstract

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.

Keywords: Diabetes Mellitus; beta cells (β Cells); induced pluripotent stem (iPS) cells; islet transplantation; stem cells.

Figure 1

Schematic representation of the mitochondrial architectural changes during somatic cell reprogramming and differentiation of iPSCs into β cells. Somatic cells undergo reprogramming to generate iPSCs, which can be re-differentiated into specialized terminal cells. This process leads to changes in the metabolic signature for mitochondria size, number, shape, fragmentation pattern (fission vs fusion), mtDNA homo/heteroplasmy, oxidative stress and metabolic pathways. Somatic cells are characterized by the presence of high number of elongated mitochondria which have a highly active OXPHOS metabolism that generates high concentrations of ROS. Reprogramming results in fission of the mitochondria that results in immature, fragmented, spherical, and perinuclear mitochondria with condensed cristae. Differentiation of iPSCs to terminal β cells requires the fusion of mitochondria to generate large numbers of enlarged mature and cristae-rick mitochondria resulting in a transition from glycolysis to OXPHOS.

Figure 2

Schematic representation of the key metabolic mechanistic pathways for energy generation in (A) iPSCs and (B) β cells. Glycolysis breaks down glucose into two molecules of pyruvate which can enter the mitochondria upon oxidative decarboxylation into acetyl CoA in the mitochondrial matrix. Acetyl-CoA then enters in the TCA cycle where it is oxidized for energy production and/ or for the generation of metabolic intermediates for fatty acid and nucleotide biosynthesis. Metabolic intermediates that arise throughout glycolysis, including glucose-6-phosphate, fructose-6-phosphate, and dihydroxyacetone phosphate (DHAP), provide the scaffolds for fatty acids and amino acids synthesis required to support the increasing biomass. Several key molecular mechanisms regulating the mitochondrial metabolic homeostasis are represented with numbers in this figure. (A) For iPSCs, three mechanisms are summzied as (1), pluripotency phenotypes (naïve vs prime)- responsible for metabolic shift between glycolysis and OXPHOS (2); mitochondrial genes affecting pluripotency genes expression to help regulating optimal glycolytic function in iPSCs; (3) Nutritional requirements facilitating glycolytic pathway for lactate production. (B) Mechanisms in human β cells include, (1) islet maturation associated genes contribute to achieve metabolic homeostasis; (2) repression of disallowed genes improves mitochondrial function and high ATP production; (3) screening mtDNA mutations will further help eliminating the risk of mitochondrial dysfunction; (4) genomic regulation of mitochondrial genes; (5) Nutritional control for ATP production and maturation; and (6) increased ROS production as a result of high mitochondrial function and respiration.

Figure 3

Schematic representation of directed differentiation of iPSC towards β cells and the metabolic shift that happens throughout the process. iPSCs can be differentiated in vitro β cells by inducing iPSCs into definitive endoderm (DE) (stage-1), which are further differentiated into primitive gut tube (PGT) (stage-2) and posterior foregut (PFT) (stage-3) before developing as pancreatic progenitors (PP) (stage-4). PPs can be further differentiated in vitro, into pancreatic endocrine precursors (PEPs) (stage-5) and lastly, into β cells (stage-6). Differentiation of iPSC towards β cells include a variety of changes for increased mitochondrial mass, mitochondrial membrane potential, ATP production, intracellular ROS level, and regulation of anaerobic and aerobic metabolic-associated genes. Briefly, differentiation to DE downregulates mitochondrial biogenesis regulators TFAM, POLG1 and POLG2, while upregulating PGC1-A, which results in increased mitochondria through upregulation of mtDNA transcription, replication, and mitochondrial membrane potential (ΔΨ). These alterations result in the maturation of the tricarboxylic acid (TCA) cycle gene transcripts. Elevated intracellular ATP levels provide energy for further differentiation and ROS generation as a result of OXPHOS. The activation of the electron transfer chain and TCA cycle promote the expression of enzymes involved in aerobic metabolism while down regulating glycolysis enzymes. Upregulation and activation of the oxidative glucose metabolism enables the activation of the triggering pathway of insulin secretion.

Figure 4

Metabolism dynamics in human primary β- cells and SC-derived β cells. The metabolic machinery in adult human β cells function optimally to sense glucose and express key metabolic enzymes that facilitate glucose-stimulated insulin secretion in response to insulin signaling and glucose transport. Glucose gets efficiently converted into pyruvate with phosphoenolpyruvate (PEP) activity which then enters mitochondrial membrane and serves as a precursor for TCA cycle respiration. Downstream enzymes converting GDP to GTP and phosphoenolpyruvate carboxykinase (PEPCK) allows continuous pyruvate flux using malate precursors which result in efficient ATP production. Increased cytosolic ATP pool then help facilitating Ka⁺ channel closure and Ca²⁺ depolarization to ensure glucose internalization and improved insulin secretion. Conversely, SC-derived β cells show anaplerotic cycling in the mitochondria. Substantial reduction in PEP activity reduces pyruvate production which further limit the mitochondrial respiratory cycle for enhanced ATP production. Further, lack of PEPCK activity results in dysfunctional mitochondria in SC-derived β cells. This overall shunt in metabolic precursors affect glucose responsive insulin production, metabolic control, and SC-β cell maturation like primary adult islets.