Adipocyte-derived Cells Research Articles

Origin of dedifferentiated adipocyte-derived cells (DFAT) during ceiling culture in an Adiponectin Cre-Recombinase mouse model.

Dedifferentiated adipose tissue-derived (DFAT) cells represent an attractive source of stem cells for tissue engineering and the potential treatment of several clinical conditions. Our objective was to determine whether DFAT cells originate from mature adipocytes and address whether contamination from the stromal vascular fraction (SVF) could be as a source for these cells. A murine adiponectin-creERT; mT/mG model was used with the excision of the cassette induced by tamoxifen injection for the cells expressing adiponectin (adipoq). This model allows distinguishing mature adipocytes (green fluorescence) from other SVF cell types (red fluorescence) based on the fluorescent protein expressed. Mature adipocytes and SVF cells were isolated from adipose tissues by collagenase digestion. Ceiling cultures were imaged by time-lapse microscopy. Confocal microscopy was used to follow cells over 21 days. Time-lapse microscopy experiments showed liposecretion occurring in mature adipocytes displaying green fluorescence. Confocal imaging allowed the identification of a heterogeneous cell population expressing green but also red fluorescence after 21 days of culture. Asymmetrical division of mature adipocytes was not observed. In conclusion, liposecretion of mature adipocytes is a phenomenon that can be observed in vitro and DFAT cells do originate from mature adipocytes. However, the population of DFAT cells is heterogenous.

Insights into the unique roles of dermal white adipose tissue (dWAT) in wound healing.

Dermal white adipose tissue (dWAT) is a newly recognized layer of adipocytes within the reticular dermis of the skin. In many mammals, this layer is clearly separated by panniculus carnosus from subcutaneous adipose tissue (sWAT). While, they concentrated around the hair shaft and follicle, sebaceous gland, and arrector pili muscle, and forms a very specific cone geometry in human. Both the anatomy and the histology indicate that dWAT has distinct development and functions. Different from sWAT, the developmental origin of dWAT shares a common precursor with dermal fibroblasts during embryogenesis. Therefore, when skin injury happens and mature adipocytes in dWAT are exposed, they may undergo lipolysis and dedifferentiate into fibroblasts to participate in wound healing as embryogenetic stage. Studies using genetic strategies to selectively ablate dermal adipocytes observed delayed revascularization and re-epithelialization in wound healing. This review specifically summarizes the hypotheses of the functions of dWAT in wound healing. First, lipolysis of dermal adipocytes could contribute to wound healing by regulating inflammatory macrophage infiltration. Second, loss of dermal adipocytes occurs at the wound edge, and adipocyte-derived cells then become ECM-producing wound bed myofibroblasts during the proliferative phase of repair. Third, mature dermal adipocytes are rich resources for adipokines and cytokines and could release them in response to injury. In addition, the dedifferentiated dermal adipocytes are more sensitive to redifferentiation protocol and could undergo expansion in infected wound. We then briefly introduce the roles of dWAT in protecting the skin from environmental challenges: production of an antimicrobial peptide against infection. In the future, we believe there may be great potential for research in these areas: (1) taking advantage of the plasticity of dermal adipocytes and manipulating them in wound healing; (2) investigating the precise mechanism of dWAT expansion in infected wound healing.

Effect of caffeine on mitochondrial biogenesis in the skeletal muscle - A narrative review.

Caffeine is one of the most widely used substances as recreational drug for performance-enhancement in sport, underpinned by a strong evidence base. Although the effects of caffeine are widely investigated within the scope of performance physiology, the molecular effects of caffeine within skeletal muscle remain unclear. Evidence from invitro and invivo models suggest that caffeine regulates the glucose metabolism in the skeletal muscle. Moreover, caffeine seems to stimulate CaMKII, PPARδ/β, AMPK and PGC1α, classical markers of exercise-adaptations, including mitochondrial biogenesis and mitochondrial content. This review summarizes evidence to suggest caffeine-effects within skeletal muscle fibers, focusing on the putative role of caffeine on mitochondrial biogenesis to explore whether caffeine supplementation might be a strategy to enhance mitochondrial biogenesis.

The distinct phenotype of primary adipocytes and adipocytes derived from stem cells of white adipose tissue as assessed by Raman and fluorescence imaging

Spectroscopy-based analysis of chemical composition of cells is a tool still scarcely used in biological sciences, although it provides unique information about the cell identity accessible in vivo and in situ. Through time-lapse spectroscopic monitoring of adipogenesis in brown and white adipose tissue-derived stem cells we have demonstrated that considerable chemical and functional changes occur along with cells differentiation and maturation, yet yielding mature adipocytes with a similar chemical composition, independent of the cellular origin (white or brown adipose tissue). However, in essence, these stem cell-derived adipocytes have a markedly different chemical composition compared to mature primary adipocytes. The consequences of this different chemical (and, hence, functional) identity have great importance in the context of selecting a suitable methodology for adipogenesis studies, particularly in obesity-related research.

Development of a physiological insulin resistance model in human stem cell-derived adipocytes.

Adipocytes are key regulators of human metabolism, and their dysfunction in insulin signaling is central to metabolic diseases including type II diabetes mellitus (T2D). However, the progression of insulin resistance into T2D is still poorly understood. This limited understanding is due, in part, to the dearth of suitable models of insulin signaling in human adipocytes. Traditionally, adipocyte models fail to recapitulate in vivo insulin signaling, possibly due to exposure to supraphysiological nutrient and hormone conditions. We developed a protocol for human pluripotent stem cell–derived adipocytes that uses physiological nutrient conditions to produce a potent insulin response comparable to in vivo adipocytes. After systematic optimization, this protocol allows robust insulin-stimulated glucose uptake and transcriptional insulin response. Furthermore, exposure of sensitized adipocytes to physiological hyperinsulinemia dampens insulin-stimulated glucose uptake and dysregulates insulin-responsive transcription. Overall, our methodology provides a novel platform for the mechanistic study of insulin signaling and resistance using human pluripotent stem cell–derived adipocytes.

Hematopoietic Stem Cell-Derived Adipocytes Modulate Adipose Tissue Cellularity, Leptin Production and Insulin Responsiveness in Female Mice.

A subpopulation of adipocytes in the major adipose depots of mice is produced from hematopoietic stem cells rather than mesenchymal progenitors that are the source of conventional white and brown/beige adipocytes. To analyze the impact of hematopoietic stem cell-derived adipocytes (HSCDAs) in the adipose niche we transplanted HSCs in which expression of a diphtheria toxin gene was under the control of the adipocyte-specific adiponectin gene promoter into irradiated wild type recipients. Thus, only adipocytes produced from HSC would be ablated while conventional white and brown adipocytes produced from mesenchymal progenitor cells would be spared. Wild type mice transplanted with HSCs from mice containing a reporter gene, but not the diphtheria toxin gene, regulated by the adiponectin gene promoter served as controls. In mice in which HSCDA production was suppressed, adipocyte size declined while adipose depot weights were unchanged and the number of conventional adipocyte progenitors significantly increased. We also measured a paradoxical increase in circulating leptin levels while physical activity was significantly decreased in the HSCDA depleted mice. Finally, insulin sensitivity was significantly reduced in HSCDA depleted mice. In contrast, loss of HSCDA production had no effect on body weight, components of energy balance, or levels of several circulating adipokines and tissue-resident inflammatory cells. These data indicate that ablation of this low-abundance subpopulation of adipocytes is associated with changes in circulating leptin levels and leptin-regulated endpoints associated with adipose tissue function. How they do so remains a mystery, but our results highlight the need for additional studies to explore the role of HSCDAs in other physiologic contexts such as obesity, metabolic dysfunction or loss of sex hormone production.

Methylglyoxal affects human mesenchymal stem cell-derived osteoblast and adipocytes differentiation in vitro

Methylglyoxal affects human mesenchymal stem cell-derived osteoblast and adipocytes differentiation in vitro

Capsaicin directly promotes adipocyte browning in the chemical compound-induced brown adipocytes converted from human dermal fibroblasts

Human brown fat is a potential therapeutic target for preventing obesity and related metabolic diseases by dissipating energy as heat through uncoupling protein 1 (UCP1). We have previously reported a method to obtain chemical compound-induced brown adipocytes (ciBAs) converted from human dermal fibroblasts under serum-free conditions. However, pharmacological responses to bioactive molecules have been poorly characterised in ciBAs. This study showed that the treatment with Capsaicin, an agonist of transient receptor potential vanilloid 1, directly activated adipocyte browning such as UCP1 expression, mitochondrial biogenesis, energy consumption rates, and glycerol recycling in ciBAs. Furthermore, genome-wide transcriptome analysis indicated that Capsaicin activated a broad range of metabolic genes including glycerol kinase and glycerol 3-phosphate dehydrogenase 1, which could be associated with the activation of glycerol recycling and triglyceride synthesis. Capsaicin also activated UCP1 expression in immortalised human brown adipocytes but inhibited its expression in mesenchymal stem cell-derived adipocytes. Altogether, ciBAs successfully reflected the direct effects of Capsaicin on adipocyte browning. These findings suggested that ciBAs could serve as a promising cell model for screening of small molecules and dietary bioactive compounds targeting human brown adipocytes.

Diurnal variations of triglyceride accumulation in mouse and bovine adipocyte-derived cell lines.

Several studies have suggested a strong interaction between the circadian clock and lipid metabolism in mammals. The circadian clock is driven by endogenous cyclic gene expression patterns, commonly referred to as clock genes, and transcription-translation negative feedback loops. Clock genes regulate the transcription of some lipid metabolism-related genes; however, the relationship between the circadian clock and triglyceride (TG) accumulation at the cellular level remains unclear. Here, we evaluated rhythms of intracellular TG accumulation levels as well as the expression of clock genes and lipid metabolism-related genes for 54 h in mouse and bovine adipose-derived cell cultures. To the best of our knowledge, this study represents the first report demonstrating that TG accumulation exhibits diurnal variations, with the pattern differing among cell types. Furthermore, we found that expression of clock genes and corresponding lipid metabolism-related genes exhibited circadian rhythms. Our results suggest that the cellular clock regulates lipid metabolism-related genes to relate circadian rhythms of TG accumulation in each cell type. We anticipate that the amount of fat stored depends on the timing of the supply of glucose-the precursor of fat. The findings of this study will contribute to the advancement of chrono-nutrition.

QS1: Creeping Fat Adipocytes Drive Intestinal Fibrosis Through Adipocyte-to-fibroblast Conversion in a Novel Model of Inflammatory Bowel Disease

Purpose:Crohn’s disease (CD) is a subtype of inflammatory bowel disease (IBD) characterized by patchy, transmural inflammation throughout the digestive tract and creeping fat formation. Thirty percent of CD patients develop strictures, eighty percent of which will require surgery. Creeping fat is associated with stricture formation, but its role in intestinal fibrosis remains unclear. Here, we present a novel surgical model of intestinal fibrosis and show by lineage tracing that creeping fat adipocytes convert to fibroblasts that contribute to fibrosis.Methods:We developed a novel surgical model of IBD that avoids the chronic use of caustic agents by creating a longitudinal, anti-mesenteric colotomy in the mouse transverse colon that is closed transversally. We performed Masson’s trichrome staining to assess collagen deposition. Finally, we performed lineage tracing of mature adipocytes in Adiponectin-Cre; mTmG mice and characterized adipocyte-derived cells by immunostaining.Results:Our surgical model mimics key features of human strictures, including the formation of creeping fat around the injury site, increased bowel wall thickness, collagen deposition, and transmural adipocyte infiltration by post-operative day (POD) 7. Immunostaining for adipocyte and fibroblast markers confirmed the presence of transmural adipocytes adjacent to fibroblasts. Lineage tracing of mature adipocytes in Adiponectin-Cre; mTmG mice revealed adipocyte-derived cells that infiltrate the wound site that lose expression of adipocyte lineage markers, gain expression of fibroblast markers, produce collagen, and respond to TGF-β signaling.Conclusions:Our novel surgical colotomy model represents a viable approach to study intestinal fibrosis and creeping fat without the long-term use of caustic agents. Lineage tracing of mature creeping fat adipocytes demonstrates that adipocytes convert to fibroblasts that infiltrate the injury site and participate in fibrotic responses. Taken together, these findings suggest that creeping fat contributes to intestinal fibrosis in part through the local conversion of adipocytes to fibroblasts.

Individual-specific functional epigenomics reveals genetic determinants of adverse metabolic effects of glucocorticoids

Glucocorticoids (GCs) are widely used as anti-inflammatory drugs, but their long-term use has severe metabolic side effects. Here, by treating multiple individual adipose stem cell-derived adipocytes and induced pluripotent stem cell-derived hepatocytes with the potent GC dexamethasone (Dex), we uncovered cell-type-specific and individual-specific GC-dependent transcriptomes and glucocorticoid receptor (GR) cistromes. Individual-specific GR binding could be traced to single-nucleotide polymorphisms (SNPs) that altered the binding motifs of GR or its cooperating factors. We also discovered another set of genetic variants that modulated Dex response through affecting chromatin accessibility or chromatin architecture. Several SNPs that altered Dex-regulated GR binding and gene expression controlled Dex-driven metabolic perturbations. Remarkably, these genetic variations were highly associated with increases in serum glucose, lipids, and body mass in subjects on GC therapy. Knowledge of the genetic variants that predispose individuals to metabolic side effects allows for a precision medicine approach to the use of clinically relevant GCs.

SIRT7 regulates lipogenesis in adipocytes through deacetylation of PPARγ2.

Aims/IntroductionPeroxisome proliferator‐activated receptor (PPAR)‐γ2 is a transcription factor crucial for regulating adipogenesis and glucose/lipid metabolism, and synthetic PPARγ ligands, such as thiazolidinediones, are effective oral medication for type 2 diabetes. Sirtuin 7 (SIRT7), a nicotinamide adenine dinucleotide‐dependent deacetylase, also controls metabolism. However, it is not known whether SIRT7 regulates the function of PPARγ2 by its deacetylation.Materials and MethodsPhysical interaction between SIRT7 and PPARγ2, the effect of SIRT7 on PPARγ2 acetylation, and the deacetylation residue targeted by SIRT7 were investigated. The effects of PPARγ2 K382 acetylation on lipid accumulation, gene expression in C3H10T1/2 cell‐derived adipocytes, and ligand‐dependent transactivation activity were also evaluated.ResultsWe demonstrated that SIRT7 binds to PPARγ2 and deacetylates PPARγ2 at K382. C3H10T1/2‐derived adipocytes expressing PPARγ2K382Q (a mimic of acetylated K) accumulated much less fat than adipocytes expressing wild‐type PPARγ2 or PPARγ2K382R (a mimic of nonacetylated K). Global gene expression analysis of adipocytes expressing PPARγ2K382Q revealed that K382Q caused the dysregulation of a set of genes involved in lipogenesis, including Srebp1c, Acaca, Fasn, and Scd1. The rosiglitazone‐dependent transcriptional activity of PPARγ2K382Q was reduced compared with that of PPARγ2K382R.ConclusionOur findings indicate that SIRT7‐dependent PPARγ2 deacetylation at K382 controls lipogenesis in adipocytes.

Caloric Restriction Alters Bone Marrow Adiposity And Energy Metabolism

BACKGROUND: Bone marrow adipose tissue (bMAT) refers to mesenchymal stem cell-derived adipocytes located within the bone marrow cavity. In adult humans, bMAT comprises ~15% of total body adiposity and is primarily located throughout the appendicular skeleton. bMAT is an extremely ‘plastic’ depot that rapidly responds to fluctuations in nutrient sensing and energetic homeostasis. Paradoxically, bMAT levels increase during both obesity and caloric restriction (CR); however, it is unclear whether the qualitative phenotype of bMAT differs under these conditions. PURPOSE: To identify obesity- and CR-induced alterations in bMAT lipid composition, transcriptional profile, and metabolic signature. METHODS: Male 6-week-old genetically obese ob/ob mice (leptin deficient) were housed at 32°C (thermoneutral) for 10 weeks. One group of mice was fed ad libitum (AL; N=9) for the study duration, while a second group was calorically restricted (CR; N=9) to ~50% of the AL group to prevent excess weight gain. Femora and tibiae were collected for lipid composition (gas chromatography), gene expression analysis (qRT-PCR), and in situ metabolic phenotyping (MPLSM-FLIM). Blood was collected for assessment of circulating analytes. RESULTS: CR mice had lower body mass and blood glucose, and higher blood corticosterone compared to AL mice. CR increased femur adipocyte density and adipocyte size compared to AL. Tibia lipid composition differed between AL and CR mice (AL>CR C16:0, C:18:1n-9, C:18:1n-7, C20:4n-6; CR >AL C14:0, C16:1n-7, and C18:2n-6). CR upregulated the expression of adipokines (e.g., Adipoq, CFD), which occurred co-incidentally with elevated levels of serum adiponectin and adipsin. Transcriptional profiling revealed differential expression of genes involved in adipogenesis, lipid metabolism, mitochondrial activity, and antioxidant response (e.g., CR>AL SCD1, FASN, LIPE, LPL, KLF15, PPARGC1a, PPARa, and SOD1). MPLSM-FLIM revealed highly heterogenic metabolic phenotypes among cells within the bone marrow cavity. CONCLUSION: CR induces functional changes in bMAT that occur independent of leptin. bMAT lies at the interface of metabolic and inflammatory signaling mechanisms, and is a potential mediator of systemic metabolic and inflammatory homeostasis during health and disease.

Mitoregulin Controls β-Oxidation in Human and Mouse Adipocytes.

SummaryWe previously discovered in mouse adipocytes an lncRNA (the homolog of human LINC00116) regulating adipogenesis that contains a highly conserved coding region. Here, we show human protein expression of a peptide within LINC00116, and demonstrate that this peptide modulates triglyceride clearance in human adipocytes by regulating lipolysis and mitochondrial β-oxidation. This gene has previously been identified as mitoregulin (MTLN). We conclude that MTLN has a regulatory role in adipocyte metabolism as demonstrated by systemic lipid phenotypes in knockout mice. We also assert its adipocyte-autonomous phenotypes in both isolated murine adipocytes as well as human stem cell-derived adipocytes. MTLN directly interacts with the β subunit of the mitochondrial trifunctional protein, an enzyme critical in the β-oxidation of long-chain fatty acids. Our human and murine models contend that MTLN could be an avenue for further therapeutic research, albeit not without caveats, for example, by promoting white adipocyte triglyceride clearance in obese subjects.

Targeting Cancer Cell Metastasis by Converting Cancer Cells into Fat.

Cancer is a systemic heterogeneous disease that can undergo several rounds of latency and activation. Malignant tumors evolve through dynamic responses to microenvironmental signals and development of resistance following therapeutic interventions. Cancer cell adaptation is required for cell survival during metastatic dissemination and outgrowth. Epithelial-mesenchymal transition (EMT) plays a major role in facilitating cell plasticity in cancer and allows cancer cells to escape chemotherapies and targeted therapies through dedifferentiation and signaling adaptation processes. In our recent study, we showed that breast cancer cells that have undergone EMT can be terminally differentiated into adipocytes using the PPARγ agonist rosiglitazone combined with the MEK inhibitor trametinib. The conversion of invasive cancer cells into adipocytes repressed primary tumor invasion and metastasis formation in mouse models of breast cancer. The transdifferentiated cancer cell-derived adipocytes were growth-arrested and lost their cellular plasticity. These results indicate the high potential of utilizing the increased cell plasticity inherent to invasive cancer cells for transdifferentiation therapy.

Caffeine exposure induces browning features in adipose tissue in vitro and in vivo

Brown adipose tissue (BAT) is able to rapidly generate heat and metabolise macronutrients, such as glucose and lipids, through activation of mitochondrial uncoupling protein 1 (UCP1). Diet can modulate UCP1 function but the capacity of individual nutrients to promote the abundance and activity of UCP1 is not well established. Caffeine consumption has been associated with loss of body weight and increased energy expenditure, but whether it can activate UCP1 is unknown. This study examined the effect of caffeine on BAT thermogenesis in vitro and in vivo. Stem cell-derived adipocytes exposed to caffeine (1 mM) showed increased UCP1 protein abundance and cell metabolism with enhanced oxygen consumption and proton leak. These functional responses were associated with browning-like structural changes in mitochondrial and lipid droplet content. Caffeine also increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression and mitochondrial biogenesis, together with a number of BAT selective and beige gene markers. In vivo, drinking coffee (but not water) stimulated the temperature of the supraclavicular region, which co-locates to the main region of BAT in adult humans, and is indicative of thermogenesis. Taken together, these results demonstrate that caffeine can promote BAT function at thermoneutrality and may have the potential to be used therapeutically in adult humans.

Exosomes secreted by mesenchymal stromal/stem cell-derived adipocytes promote breast cancer cell growth via activation of Hippo signaling pathway

ObjectiveAlthough adipocytes are the most abundant stromal cell component in breast cancer tissues, their interaction with breast cancer cells has been less investigated compared to cancer-associated fibroblasts or macrophages. Exosomes are a novel way of cell-cell communication and have been demonstrated to play an important role in various biological processes. However, to our knowledge, only a few studies have reported the effects of adipocyte exosomes on tumor development. Here, utilizing exosomes isolated from in vitro mesenchymal stromal/stem cell (MSC)-differentiated adipocytes, we systematically investigated this issue in a breast cancer model.Material and methodsExosomes were isolated from MSC-differentiated adipocytes and added to breast cancer cells MCF7. Cell proliferation was detected by MTS, and migration was analyzed by wound healing and transwell assay. An in vivo mouse xenograft model was used to evaluate MSC-differentiated adipocyte exosomes’ contribution to tumor growth. Signaling pathway activation was evaluated by western blot and immunofluorescence staining.ResultsWe found MSC-differentiated adipocyte-derived exosomes are actively incorporated by breast cancer cell MCF7 and subsequently promote MCF7 proliferation and migration as well as protect MCF7 from serum derivation or chemotherapeutic drug-induced apoptosis in vitro. In the in vivo mouse xenograft model, depletion of exosomes reduces tumor-promoting effects of adipocytes. Transcriptomic analysis of MSC-differentiated adipocyte exosome-treated MCF7 identified several activated signaling pathways, among which we confirm the Hippo signaling pathway and found a blockade of this pathway leads to a reduced growth-promoting effect of adipocyte exosomes.ConclusionTaken together, our findings provide new insights into the role of adipocyte exosomes in the tumor microenvironment.

Setting our AdipoSIGHTS on Stem Cells in Pharmacogenomics.

In a recent study published in Cell Stem Cell, Hu etal. (2019) used human adipose stem cell-derived adipocytes to demonstrate that genetic variation predicts anti-diabetic response to thiazolidinediones (TZDs) by modulating PPARγ binding.

Patient Adipose Stem Cell-Derived Adipocytes Reveal Genetic Variation that Predicts Antidiabetic Drug Response.

Thiazolidinedione drugs (TZDs) target the transcriptional activity of peroxisome proliferator activated receptor γ (PPARγ) to reverse insulin resistance in type 2 diabetes, but side effects limit their clinical use. Here, using human adipose stem cell-derived adipocytes, we demonstrate that SNPs were enriched atsites of patient-specific PPARγ binding, which correlated with the individual-specific effects of the TZD rosiglitazone (rosi) on gene expression. Rosi induction of ABCA1, which regulates cholesterol metabolism, was dependent upon SNP rs4743771, which modulated PPARγ binding by influencing the genomic occupancy of its cooperating factor, NFIA. Conversion of rs4743771 from the inactive SNP allele to the active one by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated editing rescued PPARγ binding and rosi induction of ABCA1 expression. Moreover, rs4743771 is a major determinant of undesired serum cholesterol increases in rosi-treated diabetics. These data highlight human genetic variation that impacts PPARγ genomic occupancy and patient responses to antidiabetic drugs, with implications for developing personalized therapies for metabolic disorders.

Early onset of inflammation during ontogeny of bipolar disorder: the NLRP2 inflammasome gene distinctly differentiates between patients and healthy controls in the transition between iPS cell and neural stem cell stages

Neuro-inflammation and neuronal communication are considered as mis-regulated processes in the aetiology and pathology of bipolar disorder (BD). Which and when specific signal pathways become abnormal during the ontogeny of bipolar disorder patients is unknown. To address this question, we applied induced pluripotent stem cell (iPSC) technology followed by cortical neural differentiation on adipocyte-derived cells from BD type I patients (with psychotic episodes in psychiatric history) and healthy volunteers (controls). RNA sequencing in iPSC and cortical neural stem cell (NSC) lines were used to examine alterations between the transcriptomes from BD I and control samples during transition from the pluripotent stage towards the neural developmental stage. At the iPSC stage, the most highly significant differentially expressed gene (DEG) was the NLRP2 inflammasome (P=2.66 × 10−10). Also among 42 DEGs at the NSC stage, NLRP2 showed the strongest statistical significance (P=3.07 × 10−19). In addition, we have also identified several cytoskeleton-associated genes as DEGs from the NSC stage, such as TMP2, TAGLN and ACTA2; the former two genes are recognised for the first time to be associated with BD. Our results also suggest that iPSC-derived BD-cortical NSCs carry several abnormalities in dopamine and GABA receptor canonical pathways, underlining that our in vitro BD model reflects pathology in the central nervous system. This would indicate that mis-regulated gene expression of inflammatory, neurotransmitter and cytoskeletal signalling occurs during early fetal brain development of BD I patients.