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Stem Cell Reports Jun 2023The nutritional requirements for human induced pluripotent stem cell (hiPSC) growth have not been extensively studied. Here, building on our prior work that established...
The nutritional requirements for human induced pluripotent stem cell (hiPSC) growth have not been extensively studied. Here, building on our prior work that established the suitable non-basal medium components for hiPSC growth, we develop a simplified basal medium consisting of just 39 components, demonstrating that many ingredients of DMEM/F12 are either not essential or are at suboptimal concentrations. This new basal medium along with the supplement, which we call BMEM, enhances the growth rate of hiPSCs over DMEM/F12-based media, supports derivation of multiple hiPSC lines, and allows differentiation to multiple lineages. hiPSCs cultured in BMEM consistently have enhanced expression of undifferentiated cell markers such as POU5F1 and NANOG, along with increased expression of markers of the primed state and reduced expression of markers of the naive state. This work describes titration of the nutritional requirements of human pluripotent cell culture and identifies that suitable nutrition enhances the pluripotent state.
Topics: Humans; Induced Pluripotent Stem Cells; Nutritional Requirements; Cell Culture Techniques; Cell Differentiation; Dietary Supplements
PubMed: 37315525
DOI: 10.1016/j.stemcr.2023.05.004 -
Nature Communications Jun 2023Cancers are often associated with hypoxia and metabolic reprogramming, resulting in enhanced tumor progression. Here, we aim to study breast cancer hypoxia responses,...
Cancers are often associated with hypoxia and metabolic reprogramming, resulting in enhanced tumor progression. Here, we aim to study breast cancer hypoxia responses, focusing on secreted proteins from low-grade (luminal-like) and high-grade (basal-like) cell lines before and after hypoxia. We examine the overlap between proteomics data from secretome analysis and laser microdissected human breast cancer stroma, and we identify a 33-protein stromal-based hypoxia profile (33P) capturing differences between luminal-like and basal-like tumors. The 33P signature is associated with metabolic differences and other adaptations following hypoxia. We observe that mRNA values for 33P predict patient survival independently of molecular subtypes and basic prognostic factors, also among low-grade luminal-like tumors. We find a significant prognostic interaction between 33P and radiation therapy.
Topics: Humans; Female; Breast Neoplasms; Proteome; Gene Expression Profiling; Cell Line, Tumor; Hypoxia; Gene Expression Regulation, Neoplastic
PubMed: 37349288
DOI: 10.1038/s41467-023-39287-7 -
ELife Sep 2023The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to...
The primary cilium plays important roles in regulating cell differentiation, signal transduction, and tissue organization. Dysfunction of the primary cilium can lead to ciliopathies and cancer. The formation and organization of the primary cilium are highly associated with cell polarity proteins, such as the apical polarity protein CRB3. However, the molecular mechanisms by which CRB3 regulates ciliogenesis and the location of CRB3 remain unknown. Here, we show that CRB3, as a navigator, regulates vesicle trafficking in γ-tubulin ring complex (γTuRC) assembly during ciliogenesis and cilium-related Hh and Wnt signaling pathways in tumorigenesis. knockout mice display severe defects of the primary cilium in the mammary ductal lumen and renal tubule, while mammary epithelial-specific knockout mice exhibit the promotion of ductal epithelial hyperplasia and tumorigenesis. CRB3 is essential for lumen formation and ciliary assembly in the mammary epithelium. We demonstrate that CRB3 localizes to the basal body and that CRB3 trafficking is mediated by Rab11-positive endosomes. Significantly, CRB3 interacts with Rab11 to navigate GCP6/Rab11 trafficking vesicles to CEP290, resulting in intact γTuRC assembly. In addition, CRB3-depleted cells are unresponsive to the activation of the Hh signaling pathway, while CRB3 regulates the Wnt signaling pathway. Therefore, our studies reveal the molecular mechanisms by which CRB3 recognizes Rab11-positive endosomes to facilitate ciliogenesis and regulates cilium-related signaling pathways in tumorigenesis.
Topics: Animals; Mice; Basal Bodies; Carcinogenesis; Cell Differentiation; Cell Transformation, Neoplastic; Hyperplasia; Microtubule-Organizing Center
PubMed: 37737843
DOI: 10.7554/eLife.86689 -
The Journal of Investigative Dermatology Jul 2023Chromatin landscape and regulatory networks are determinants in lineage specification and differentiation. To define the temporospatial differentiation axis in murine...
Chromatin landscape and regulatory networks are determinants in lineage specification and differentiation. To define the temporospatial differentiation axis in murine epidermal cells in vivo, we generated datasets profiling expression dynamics (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin using sequencing), architecture (Hi-C), and histone modifications (chromatin immunoprecipitation followed by sequencing) in the epidermis. We show that many differentially regulated genes are suppressed during the differentiation process, with superenhancers controlling differentiation-specific epigenomic changes. Our data shows the relevance of the Dlx/Klf/Grhl combinatorial regulatory network in maintaining correct temporospatial gene expression during epidermal differentiation. We determined differential open compartments, topologically associating domain score, and looping in the basal cell and suprabasal cell epidermal fractions, with the evolutionarily conserved epidermal differentiation complex region showing distinct suprabasal cell-specific topologically associating domain and loop formation that coincided with superenhancer sites. Overall, our study provides a global genome-wide resource of chromatin dynamics that define unrecognized regulatory networks and the epigenetic control of Dlx3-bound superenhancer elements during epidermal differentiation.
Topics: Mice; Animals; Chromatin; Transcription Factors; Cell Differentiation; Epidermis; Epidermal Cells
PubMed: 36708949
DOI: 10.1016/j.jid.2022.12.020 -
The Journal of Nutritional Biochemistry Oct 2023Omega-3 fatty acids (w-3 FA) have anti-inflammatory effects and improve mitochondrial function. Nonetheless, little is known about their effect on mitochondrial...
Omega-3 fatty acids (w-3 FA) have anti-inflammatory effects and improve mitochondrial function. Nonetheless, little is known about their effect on mitochondrial bioenergetics of peripheral blood mononuclear cells (PBMCs) in individuals with obesity. Thus, this study aimed to determine the mitochondrial bioenergetics status and cell subset composition of PBMCs during obesity, before and after 1 month supplementation with w-3 FA. We performed a case-control study with twelve women with normal BMI (lean group) and 19 with grade 2 obesity (obese group), followed by a before-after prospective study where twelve subjects with obesity received a 1 month intervention with 5.25 g of w-3 FA (3.5 g eicosapentaenoic (EPA) and 1.75 g docosahexaenoic (DHA) acids), and obtained PBMCs from all participants. Mitochondrial bioenergetic markers, including basal and ATP-production associated respiration, proton leak, and nonmitochondrial respiration, were higher in PBMCs from the obese group vs. the lean group. The bioenergetic health index (BHI), a marker of mitochondrial function, was lower in the obese vs. the lean group. In addition, Th1, Th2, Th17, CD4+ Tregs, CD8+ Tregs, and Bregs, M1 monocytes and pDCreg cells were higher in PBMCs from the obese group vs. the lean group. The w-3 FA intervention improved mitochondrial function, mainly by decreasing nonmitochondrial respiration and increasing the reserve respiratory capacity and BHI. The intervention also reduced circulating pro-inflammatory and anti-inflammatory lymphocyte and monocytes subsets in individuals with obesity. The mitochondrial dysfunction of PBMCs and the higher proportion of peripheral pro-inflammatory and anti-inflammatory immune cells in subjects with obesity, improved with 1 month supplementation with EPA and DHA.
Topics: Humans; Female; Leukocytes, Mononuclear; Case-Control Studies; Prospective Studies; Docosahexaenoic Acids; Fatty Acids, Omega-3; Obesity; Inflammation; Mitochondria; Dietary Supplements; Eicosapentaenoic Acid; Fatty Acids
PubMed: 37437746
DOI: 10.1016/j.jnutbio.2023.109415 -
Scientific Reports Oct 2023The nasal and bronchial epithelium are unified parts of the respiratory tract that are affected in the monogenic disorder cystic fibrosis (CF). Recent studies have...
The nasal and bronchial epithelium are unified parts of the respiratory tract that are affected in the monogenic disorder cystic fibrosis (CF). Recent studies have uncovered that nasal and bronchial tissues exhibit intrinsic variability, including differences in mucociliary cell composition and expression of unique transcriptional regulatory proteins which relate to germ layer origin. In the present study, we explored whether intrinsic differences between nasal and bronchial epithelial cells persist in cell cultures and affect epithelial cell functioning in CF. Comparison of air-liquid interface (ALI) differentiated epithelial cells from subjects with CF revealed distinct mucociliary differentiation states of nasal and bronchial cultures. Moreover, using RNA sequencing we identified cell type-specific signature transcription factors in differentiated nasal and bronchial epithelial cells, some of which were already poised for expression in basal progenitor cells as evidenced by ATAC sequencing. Analysis of differentiated nasal and bronchial epithelial 3D organoids revealed distinct capacities for fluid secretion, which was linked to differences in ciliated cell differentiation. In conclusion, we show that unique phenotypical and functional features of nasal and bronchial epithelial cells persist in cell culture models, which can be further used to investigate the effects of tissue-specific features on upper and lower respiratory disease development in CF.
Topics: Humans; Cystic Fibrosis; Cells, Cultured; Respiratory Mucosa; Nose; Epithelial Cells; Cystic Fibrosis Transmembrane Conductance Regulator
PubMed: 37903789
DOI: 10.1038/s41598-023-45201-4 -
Developmental Cell Dec 2023The extrahepatic branches of the biliary tree have glands that connect to the surface epithelium through narrow pits. The duct epithelia undergo homeostatic renewal, yet...
The extrahepatic branches of the biliary tree have glands that connect to the surface epithelium through narrow pits. The duct epithelia undergo homeostatic renewal, yet the identity and multiplicity of cells that maintain this tissue is unknown. Using marker-free and targeted clonal fate mapping in mice, we provide evidence that the extrahepatic bile duct is compartmentalized. Pit cholangiocytes of extramural glands renewed the surface epithelium, whereas basally oriented cholangiocytes maintained the gland itself. In contrast, basally positioned cholangiocytes replenished the surface epithelium in mural glands. Single-cell sequencing identified genes enriched in the base and surface epithelial populations, with trajectory analysis showing graded gene expression between these compartments. Epithelia were plastic, changing cellular identity upon fasting and refeeding. Gain of canonical Wnt signaling caused basal cell expansion, gastric chief cell marker expression, and a decrease in surface epithelial markers. Our results identify the cellular hierarchy governing extrahepatic biliary epithelial renewal.
Topics: Animals; Mice; Biliary Tract; Bile Ducts, Extrahepatic; Epithelium; Epithelial Cells; Cell Proliferation
PubMed: 37909044
DOI: 10.1016/j.devcel.2023.10.004 -
Journal of Cell Science Dec 2023The centrosome is a non-membrane-bound organelle that is conserved across most animal cells and serves various functions throughout the cell cycle. In dividing cells,... (Review)
Review
The centrosome is a non-membrane-bound organelle that is conserved across most animal cells and serves various functions throughout the cell cycle. In dividing cells, the centrosome is known as the spindle pole and nucleates a robust microtubule spindle to separate genetic material equally into two daughter cells. In non-dividing cells, the mother centriole, a substructure of the centrosome, matures into a basal body and nucleates cilia, which acts as a signal-transducing antenna. The functions of centrosomes and their substructures are important for embryonic development and have been studied extensively using in vitro mammalian cell culture or in vivo using invertebrate models. However, there are considerable differences in the composition and functions of centrosomes during different aspects of vertebrate development, and these are less studied. In this Review, we discuss the roles played by centrosomes, highlighting conserved and divergent features across species, particularly during fertilization and embryonic development.
Topics: Animals; Centrosome; Centrioles; Cell Cycle; Microtubules; Fertilization; Mammals
PubMed: 38038054
DOI: 10.1242/jcs.261387 -
Cellular and Molecular Life Sciences :... Aug 2023Huntington's disease (HD) is an incurable inherited brain disorder characterised by massive degeneration of striatal neurons, which correlates with abnormal accumulation...
Huntington's disease (HD) is an incurable inherited brain disorder characterised by massive degeneration of striatal neurons, which correlates with abnormal accumulation of misfolded mutant huntingtin (mHTT) protein. Research on HD has been hampered by the inability to study early dysfunction and progressive degeneration of human striatal neurons in vivo. To investigate human pathogenesis in a physiologically relevant context, we transplanted human pluripotent stem cell-derived neural progenitor cells (hNPCs) from control and HD patients into the striatum of new-born mice. Most hNPCs differentiated into striatal neurons that projected to their target areas and established synaptic connexions within the host basal ganglia circuitry. Remarkably, HD human striatal neurons first developed soluble forms of mHTT, which primarily targeted endoplasmic reticulum, mitochondria and nuclear membrane to cause structural alterations. Furthermore, HD human cells secreted extracellular vesicles containing mHTT monomers and oligomers, which were internalised by non-mutated mouse striatal neurons triggering cell death. We conclude that interaction of mHTT soluble forms with key cellular organelles initially drives disease progression in HD patients and their transmission through exosomes contributes to spread the disease in a non-cell autonomous manner.
Topics: Humans; Animals; Mice; Huntington Disease; Neurons; Neural Stem Cells; Corpus Striatum; Cell Differentiation; Huntingtin Protein; Disease Models, Animal
PubMed: 37535170
DOI: 10.1007/s00018-023-04882-w -
Human Molecular Genetics Sep 2023Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene have been identified as one of the most common genetic causes of Parkinson's disease (PD). The LRRK2...
Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene have been identified as one of the most common genetic causes of Parkinson's disease (PD). The LRRK2 PD-associated mutations LRRK2G2019S and LRRK2R1441C, located in the kinase domain and in the ROC-COR domain, respectively, have been demonstrated to impair mitochondrial function. Here, we sought to further our understanding of mitochondrial health and mitophagy by integrating data from LRRK2R1441C rat primary cortical and human induced pluripotent stem cell-derived dopamine (iPSC-DA) neuronal cultures as models of PD. We found that LRRK2R1441C neurons exhibit decreased mitochondrial membrane potential, impaired mitochondrial function and decreased basal mitophagy levels. Mitochondrial morphology was altered in LRRK2R1441C iPSC-DA but not in cortical neuronal cultures or aged striatal tissue, indicating a cell-type-specific phenotype. Additionally, LRRK2R1441C but not LRRK2G2019S neurons demonstrated decreased levels of the mitophagy marker pS65Ub in response to mitochondrial damage, which could disrupt degradation of damaged mitochondria. This impaired mitophagy activation and mitochondrial function were not corrected by the LRRK2 inhibitor MLi-2 in LRRK2R1441C iPSC-DA neuronal cultures. Furthermore, we demonstrate LRRK2 interaction with MIRO1, a protein necessary to stabilize and to anchor mitochondria for transport, occurs at mitochondria, in a genotype-independent manner. Despite this, we found that degradation of MIRO1 was impaired in LRRK2R1441C cultures upon induced mitochondrial damage, suggesting a divergent mechanism from the LRRK2G2019S mutation.
Topics: Humans; Rats; Animals; Aged; Parkinson Disease; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Mitophagy; Induced Pluripotent Stem Cells; Mutation; Mitochondria
PubMed: 37384414
DOI: 10.1093/hmg/ddad102