Serum-assisted metabolic priming enables robust structural maturation of hiPSC-cardiomyocytes in 2D and 3D systems.

The purpose of this study was to examine the induction of epithelial to mesenchymal transition (EMT) in a prostate cancer cell line by measuring classical biomarker expression in three-dimensional (3D) spheroid cultures compared to traditional 2D monolayers in an effort to develop a more biologically relevant assay. Using Ultra-Low Attachment (ULA) microplates, we grew spheroids from a human prostate cancer cell line (DU 145). Numerous studies have implicated a role for EMT in carcinoma invasion and metastasis. EMT is characterized by rearrangement of the extracellular matrix (ECM) and differential regulation of ECM proteins. We induced EMT using TGF-beta and phorbol-12-myristate-13-acetate (PMA) and compared expression levels of specific biomarkers, such as E-cadherin, fibronectin, and IL-6, using AlphaLISA and LANCE (TR-FRET) assay technologies. We confirmed that treatment of DU 145 cells with TGF-beta is sufficient for inducing changes in both EMT biomarker expression and characteristic cellular morphology in monolayer cultures. However, in 3D spheroid cultures, we observed only a partial EMT response to the same TGF-beta treatment as demonstrated by changes in the expected biomarker expression pattern. Using the small molecule, PMA, we see significant differences in the levels of IL-6 secretion after EMT induction between cells grown in monolayer and those grown in spheroids. Cellular proliferation, growth and vitality were assessed using ATPlite luminescence assays and confocal microscopy of live-stained cells with a high content imaging system. Though we observe increased proliferation in monolayer cultures compared to 3D spheroids, the changes observed in protein expression patterns cannot be sufficiently explained by differences in cell number or viability. These data illustrate the differences in protein expression levels and in cellular tolerance for compound treatment between a human prostate cancer cell line grown in monolayers and those same cells grown in 3D spheroids. Citation Format: Jen Carlstrom, Jeanine M. Hinterneder, Lindsay Nelson, Stephen Hurt. Comparison of EMT biomarker expression in 2D monolayer and 3D spheroid cultures in a prostate cancer cell model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5793. doi:10.1158/1538-7445.AM2017-5793

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are cells with promising applications. However, their immaturity has restricted their use in cell therapy, disease modeling, and other studies. Therefore, the current study focused on inducing the maturation of CMs. We supplemented hiPSC-CMs with fatty acids (FAs) to promote their phenotypic maturity. Proteomic sequencing was performed to identify regulators critical for promoting the maturation of hiPSC-CMs. AKAP1 was found to be significantly increased in FA-treated hiPSC-CMs, and the results were verified. Therefore, we inhibited AKAP1 expression in the FA-treated cells and analyzed the outcomes. FA supplementation promoted the morphological and functional maturation of the hiPSC-CMs, which was accompanied by the development of a mitochondrial network. Proteomic analysis results revealed that AKAP1 expression was significantly higher in FA-treated hiPSC-CMs than in control cells. In addition, increased phosphorylation of the mitochondrial dynamin Drp1 and an increased mitochondrial fusion rate were found in FA-treated hiPSC-CMs. After AKAP1 was knocked down, the level of DRP1 phosphorylation in the cell was decreased, and the mitochondrial fusion rate was reduced. FA supplementation effectively promoted the maturation of hiPSC-CMs, and in these cells, AKAP1 regulated mitochondrial dynamics, possibly playing a significant role.

Cystic fibrosis (CF) is associated with fatty acid alterations characterized by low linoleic and docosahexaenoic acid. It is not clear whether these fatty acid alterations are directly linked to cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction or result from nutrient malabsorption. We hypothesized that if fatty acid alterations are a result of CFTR dysfunction, those alterations should be demonstrable in CF cell culture models. Two CF airway epithelial cell lines were used: 16HBE, sense and antisense CFTR cells, and C38/IB3-1 cells. Wild-type (WT) and CF cells were cultured in 10% fetal bovine serum (FBS) or 10% horse serum. Fatty acid levels were analyzed by GC-MS. Culture of both WT and CF cells in FBS resulted in very low linoleic acid levels. When cells were cultured in horse serum containing concentrations of linoleic acid matching those found in human plasma, physiological levels of linoleic acid were obtained and fatty acid alterations characteristic of CF tissues were then evident in CF compared with WT cells. Kinetic studies with radiolabeled linoleic acid demonstrated in CF cells increased conversion to longer and more-desaturated fatty acids such as arachidonic acid. In conclusion, these data demonstrate that CFTR dysfunction is associated with altered fatty acid metabolism in cultured airway epithelial cells.

Three-dimensional spheroids of dedifferentiated fat cells enhance bone regeneration.

The two-dimensional (2D) monolayer culture as a conventional method has been widely applied in molecular biology fields, but it has limited capability to recapitulate real cell environments, being prone to misinterpretation with poor prediction of in vivo behavior. Recently, the three-dimensional (3D) spheroid culture has been studied extensively. Spheroids are self-assembled cell aggregates that have biomimicry capabilities. The behavior of thyroid cancer under the 3D spheroid culture environment has been studied; however, there are no reports regarding differences in the degree of thyroid cancer cell differentiation under 2D and 3D culture conditions. This study investigated the expression of thyroid differentiation proteins related to iodide-metabolizing mechanisms in thyroid cancer cells under different culture conditions. Four thyroid cancer cell lines and one thyroid follicular epithelial cell line were grown in adherent 2D cell culture and 3D spheroid culture with agarose-coated plates. We observed changes in proliferation, hypoxia, extracellular matrix (ECM), cytoskeleton, thyroid-specific proteins, and thyroid transcription factors. All cell lines were successfully established in the spheroid following cell aggregation. Proliferation considerably decreased, while hypoxia-inducible factor 1-α(HIF1-α) was promoted in 3D spheroids; moreover, 3D spheroids with thyroid cancers showed diminished thyroid differentiation markers, but thyroid follicular epithelial cells revealed either a maintenance or weak decline of protein expression. We verified that the 3D spheroid culture environment can be similar to in vivo conditions because of its alterations in numerous cellular and functional activities, including morphology, cellular proliferation, viability, hypoxia, ECM, cytoskeleton, and thyroid differentiation, compared to the conventional 2D monolayer culture environment. An in vitro experimental study using 3D spheroid culture is ideal for the faster discovery of new drugs.

Cytochrome P450 (CYP) 3A4 induction is an important cause of drug-drug interactions, making early identification of drug candidates with CYP3A4 induction liability in drug development a prerequisite. Here, we present three-dimensional (3D) spheroid cultures of primary human hepatocytes (PHHs) as a novel CYP3A4 induction screening model. Screening of 25 drugs (12 known CYP3A4 inducers in vivo and 13 negative controls) at physiologically relevant concentrations revealed a 100% sensitivity and 100% specificity of the system. Three of the in vivo CYP3A4 inducers displayed much higher CYP3A4 induction capacity in 3D spheroid cultures as compared with in two-dimensional (2D) monolayer cultures. Among those, we identified AZD1208, a proviral integration site for Moloney murine leukemia virus (PIM) kinase inhibitor terminated in phase I of development due to unexpected CYP3A4 autoinduction, as a CYP3A4 inducer only active in 3D spheroids but not in 2D monolayer cultures. Gene knockdown experiments revealed that AZD1208 requires pregnane X receptor (PXR) to induce CYP3A4. Rifampicin requires solely PXR to induce CYP3A4 and CYP2B6, while phenobarbital-mediated induction of these CYPs did not show absolute dependency on either PXR or constitutive androstane receptor (CAR), suggesting its ability to switch nuclear receptor activation. Mechanistic studies into AZD1208 uncovered an involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in CYP3A4 induction that is sensitive to the culture format used, as revealed by its inhibition of ERK1/2 Tyrosine 204 phosphorylation and sensitivity to epidermal growth factor (EGF) pressure. In line, we also identified lapatinib, a dual epidermal growth factor receptor/human epidermal growth factor receptor 2 (EGFR/HER2) inhibitor, as another CYP3A4 inducer only active in 3D spheroid culture. Our findings offer insights into the pathways involved in CYP3A4 induction and suggest PHH spheroids for preclinical CYP3A4 induction screening.

Three-dimensional spheroid culture promotes odonto/osteoblastic differentiation of dental pulp cells

SIRT3 promotes metabolic maturation of human iPSC-derived cardiomyocytes via OPA1-controlled mitochondrial dynamics

Nitroalkene derivatives of linoleic acid (LNO2) and oleic acid (OA-NO2) are present; however, their biological functions remain to be fully defined. Herein, we report that LNO2 and OA-NO2 inhibit lipopolysaccharide-induced secretion of proinflammatory cytokines in macrophages independent of nitric oxide formation, peroxisome proliferator-activated receptor-gamma activation, or induction of heme oxygenase-1 expression. The electrophilic nature of fatty acid nitroalkene derivatives resulted in alkylation of recombinant NF-kappaB p65 protein in vitro and a similar reaction with p65 in intact macrophages. The nitroalkylation of p65 by fatty acid nitroalkene derivatives inhibited DNA binding activity and repressed NF-kappaB-dependent target gene expression. Moreover, nitroalkenes inhibited endothelial tumor necrosis factor-alpha-induced vascular cell adhesion molecule 1 expression and monocyte rolling and adhesion. These observations indicate that nitroalkenes such as LNO2 and OA-NO2, derived from reactions of unsaturated fatty acids and oxides of nitrogen, are a class of endogenous anti-inflammatory mediators.

Single-cell analysis reveals the purification and maturation effects of glucose starvation in hiPSC-CMs

BackgroundAlthough human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel Kir2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking.MethodsiPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs).ResultsWe showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of Kir2.1 and current density of Kir2.1-encoded IK1. The larger IK1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by IK1 blocker BaCl2. KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca2+ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction.ConclusionsOverexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca2+ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine.

Mass production of exosomes is a prerequisite for their commercial utilization. This study investigated whether three-dimensional (3D) spheroid culture of mesenchymal stem cells (MSCs) could improve the production efficiency of exosomes and if so, what was the mechanism involved. We adopted two models of 3D spheroid culture using the hanging-drop (3D-HD) and poly(2-hydroxyethyl methacrylate) (poly-HEMA) coating methods (3D-PH). The efficiency of exosome production from MSCs in the 3D spheroids was compared with that of monolayer culture in various conditions. We then investigated the mechanism of the 3D spheroid culture-induced increase in exosome production. The 3D-HD formed a single larger spheroid, while the 3D-PH formed multiple smaller ones. However, MSCs cultured on both types of spheroids produced significantly more exosomes than those cultured in conventional monolayer culture (2D). We then investigated the cause of the increased exosome production in terms of hypoxia within the 3D spheroids, high cell density, and non-adherent cell morphology. With increasing spheroid size, the efficiency of exosome production was the largest with the least amount of cells in both 3D-HD and 3D-PH. An increase in cell density in 2D culture (2D-H) was less efficient in exosome production than the conventional, lower cell density, 2D culture. Finally, when cells were plated at normal density on the poly-HEMA coated spheroids (3D-N-PH); they formed small aggregates of less than 10 cells and still produced more exosomes than those in the 2D culture when plated at the same density. We also found that the expression of F-actin was markedly reduced in the 3D-N-PH culture. These results suggested that 3D spheroid culture produces more exosomes than 2D culture and the non-adherent round cell morphology itself might be a causative factor. The result of the present study could provide useful information to develop an optimal process for the mass production of exosomes.

SummaryWhen cultured under typical conditions, human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are structurally and functionally immature. We have previously demonstrated that culture of hiPSC-CMs in maturation medium containing fatty acids, in combination with culture on micropatterned surfaces, produces cells that demonstrate a more mature phenotype compared to standard approaches. Here, we show in detail the steps needed to produce mature hiPSC-CMs. Compared with many approaches, our protocol is relatively simple and can be easily adapted to new laboratories.For complete details on the use and execution of this protocol, please refer to Knight et al. (2021).

Saturated and monounsaturated fatty acids are the most abundant fatty acid species in mammalian organisms, and their distribution is regulated by stearoyl-CoA desaturase, the enzyme that converts saturated into monounsaturated fatty acids. A positive correlation between high monounsaturated fatty acid levels and neoplastic transformation has been reported, but little is still known about the regulation of stearoyl-CoA desaturase in cell proliferation and apoptosis, as well as in cancer development. Here we report that simian virus 40-transformed human lung fibroblasts bearing a knockdown of human stearoyl-CoA desaturase by stable antisense cDNA transfection (hSCDas cells) showed a considerable reduction in monounsaturated fatty acids, cholesterol, and phospholipid synthesis, compared with empty vector transfected-simian virus 40 cell line (control cells). hSCDas cells also exhibited high cellular levels of saturated free fatty acids and triacylglycerol. Interestingly, stearoyl-CoA desaturase-depleted cells exhibited a dramatic decrease in proliferation rate and abolition of anchorage-independent growth. Prolonged exposure to exogenous oleic acid did not reverse either the slower proliferation or loss of anchorage-independent growth of hSCDas cells, suggesting that endogenous synthesis of monounsaturated fatty acids is essential for rapid cell replication and invasiveness, two hallmarks of neoplastic transformation. Moreover, apoptosis was increased in hSCDas cells in a ceramide-independent manner. Finally, stearoyl-CoA desaturase-deficient cells were more sensitive to palmitic acid-induced apoptosis compared with control cells. Our data suggest that, by globally regulating lipid metabolism, stearoyl-CoA desaturase activity modulates cell proliferation and survival and emphasize the important role of endogenously synthesized monounsaturated fatty acids in sustaining the neoplastic phenotype of transformed cells.

Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ECR (European research council) and ReNew (the Novo Nordisk Foundation Center for Stem Cell Medicine supported by Novo Nordisk Foundation grants) Introduction Cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs) are functionally immature and this limits the application of these cells to cardiovascular research. Maturation can be improved by aggregating hiPSC-CMs into three-dimensional (3D) microtissues together with hiPSC-cardiac fibroblasts (hiPSC-CFs) and hiPSC-endothelial cells (hiPSC-ECs). These cardiac microtissues showed improved structural (increase of sarcomere length and organisation) and functional (more mature action potential profile) properties. This effect was only observed when the cardiac microtissues contained hiPSC-CFs. Since connexin 43 (CX43), the most ubiquitous connexin isoform in the heart, was significantly upregulated in hiPSC-CMs from cardiac microtissues, we hypothesise that mechanisms underlying hiPSC-CMs maturation involves the coupling of hiPSC-CMs with hiPSC-CFs through this protein. Purpose The aim of this study is to unravel the role of CX43 in the maturation of hiPSC-CMs in the context of 3D multicellular cardiac tissues. Methods We used the CRISPR/Cas9 technology to generate a CX43 knock-out (KO) hiPSC line. We designed two sgRNAs annealing to exon 2 of GJA1 gene, encoding CX43; indels generated by the non-homologous end joining after the Cas9 double strand break will result in a non-functional protein. Cells were differentiated in vitro into CMs, epicardial cells (EPI) and then CFs and characterized for the expression of cell-specific markers and compared with the wild-type parental control cells. Results and conclusions We screened 31 hiPSC clones and selected those in which both GJA1 alleles contained a deletion. Copy number variation analysis by ddPCR of the flanking regions confirmed that the deletion was specific and confined. The lack of GJA1 expression in the KO clones was confirmed by qPCR, while the lack of the CX43 protein was confirmed by immunofluorescence and Western Blot. This did not affect CX43-KO hiPSCs pluripotency state shown by the presence of pluripotent markers such as NANOG, OCT3/4, SSEA4. Additionally, CX43-KO hiPSCs were able to differentiate into the three germ layers. CX43-KO hiPSCs were successfully differentiated into hiPSC-CMs, -EPI, and -CFs. CX43-KO hiPSC-CMs and controls showed comparable gene expression of cardiac troponin T. Since immunofluorescent staining showed that CX43-KO hiPSC-EPI expressed the epicardial markers WT1 and ZO1, we continued to differentiate them into hiPSC-CFs, which expressed markers such as VIM and COL1A1, similar to the control. Differentiated CX43-KO hiPSC-CMs and CFs will be assembled into cardiac microtissues. Functional and structural assays that include patch-clamp and immunofluorescence will be performed on these microtissue to assess the effect of the CX43 KO on the maturation of hiPSC-CMs. Our results show that the lack of CX43 expression does not impede hiPSCs to differentiate into various cell types originating from the mesoderm germ layer.