Differentiation of ciliated human midbrain-derived LUHMES neurons

ABSTRACTMany human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary, and an easy-to-maintain, ciliated human neuronal cell model is absent. The Lund human mesencephalic (LUHMES) cell line is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as postmitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The sonic hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-sequencing timecourse analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain. In LUHMES cells, neuronal cilia biology can be investigated from proliferation through differentiation to mature neurons.

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Differentiation of ciliated human midbrain-derived LUHMES neurons

10.1101/2020.06.04.134478 ◽

2020 ◽

Author(s):

Gilbert Lauter ◽

Andrea Coschiera ◽

Masahito Yoshihara ◽

Debora Sugiaman-Trapman ◽

Sini Ezer ◽

...

Keyword(s):

Regulatory Networks ◽

Time Course ◽

Developmental Stages ◽

Cell Model ◽

Neuronal Cell ◽

Cell Types ◽

Neuronal Cilia ◽

Human Neuronal Cell ◽

Mature Neurons

AbstractMany human cell types are ciliated, including neural progenitors and differentiated neurons. Ciliopathies are characterized by defective cilia and comprise various disease states, including brain phenotypes, where the underlying biological pathways are largely unknown. Our understanding of neuronal cilia is rudimentary and an easy-to-maintain, ciliated human neuronal cell model is missing.LUHMES is a ciliated neuronal cell line derived from human fetal mesencephalon. LUHMES cells can easily be maintained and differentiated into mature, functional neurons within one week. They have a single primary cilium as proliferating progenitor cells and as post-mitotic, differentiating neurons. These developmental stages are completely separable within one day of culture condition change. The Sonic Hedgehog (SHH) signaling pathway is active in differentiating LUHMES neurons. RNA-seq time course analyses reveal molecular pathways and gene-regulatory networks critical for ciliogenesis and axon outgrowth at the interface between progenitor cell proliferation, polarization and neuronal differentiation. Gene expression dynamics of cultured LUHMES neurons faithfully mimic the corresponding in vivo dynamics of human fetal midbrain.In LUHMES, neuronal cilia biology can be investigated along a complete timeline: from proliferation through differentiation to mature neurons.Summary StatementWith LUHMES, a ciliated human neuronal cell model, the underlying “neurobiology” of cilia and ciliopathies can be investigated along a complete time line: from proliferation through differentiation to mature neurons.

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Ultrastructural Study of a differentiating human colon carcinoma cell line

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158583 ◽

1990 ◽

Vol 48 (3) ◽

pp. 208-209

Author(s):

Sylvie Polak-Charcon ◽

Mehrdad Hekmati ◽

Yehuda Ben Shaul

Keyword(s):

Cell Line ◽

Morphological Changes ◽

Secretory Granules ◽

Human Colon ◽

Cell Types ◽

Culture Conditions ◽

Morphological Evidence ◽

Human Colon Carcinoma Cell ◽

Colon Cell

The epithelium of normal human colon mucosa “in vivo” exhibits a gradual pattern of differentiation as undifferentiated stem cells from the base of the crypt of “lieberkuhn” rapidly divide, differentiate and migrate toward the free surface. The major differentiated cell type of the intestine observed are: absorptive cells displaying brush border, goblet cells containing mucous granules, Paneth and endocrine cells containing dense secretory granules. These different cell types are also found in the intestine of the 13-14 week old embryo.We present here morphological evidence showing that HT29, an adenocarcinoma of the human colon cell line, can differentiate into various cell types by changing the growth and culture conditions and mimic morphological changes found during development of the intestine in the human embryo.HT29 cells grown in tissue-culture dishes in DMEM and 10% FCS form at late confluence a multilayer of morphologically undifferentiated cell culture covered with irregular microvilli, and devoid of tight junctions (Figs 1-3).

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The landscape of alternative polyadenylation in single cells of the developing mouse embryo

Nature Communications ◽

10.1038/s41467-021-25388-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Vikram Agarwal ◽

Sereno Lopez-Darwin ◽

David R. Kelley ◽

Jay Shendure

Keyword(s):

Rna Binding ◽

Developmental Stages ◽

Single Cells ◽

Neuronal Cell ◽

Alternative Polyadenylation ◽

Cell Types ◽

Untranslated Regions ◽

Mammalian Development ◽

Translation Rate ◽

Dynamic Landscape

Abstract3′ untranslated regions (3′ UTRs) post-transcriptionally regulate mRNA stability, localization, and translation rate. While 3′-UTR isoforms have been globally quantified in limited cell types using bulk measurements, their differential usage among cell types during mammalian development remains poorly characterized. In this study, we examine a dataset comprising ~2 million nuclei spanning E9.5–E13.5 of mouse embryonic development to quantify transcriptome-wide changes in alternative polyadenylation (APA). We observe a global lengthening of 3′ UTRs across embryonic stages in all cell types, although we detect shorter 3′ UTRs in hematopoietic lineages and longer 3′ UTRs in neuronal cell types within each stage. An analysis of RNA-binding protein (RBP) dynamics identifies ELAV-like family members, which are concomitantly induced in neuronal lineages and developmental stages experiencing 3′-UTR lengthening, as putative regulators of APA. By measuring 3′-UTR isoforms in an expansive single cell dataset, our work provides a transcriptome-wide and organism-wide map of the dynamic landscape of alternative polyadenylation during mammalian organogenesis.

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Effects of taurolithocholate, a Ca2+-mobilizing agent, on cell Ca2+ in rat hepatocytes, human platelets and neuroblastoma NG108-15 cell line

Biochemical Journal ◽

10.1042/bj2730153 ◽

1991 ◽

Vol 273 (1) ◽

pp. 153-160 ◽

Cited By ~ 10

Author(s):

J F Coquil ◽

B Berthon ◽

N Chomiki ◽

L Combettes ◽

P Jourdon ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Bile Acid ◽

Cell Line ◽

Rat Liver ◽

Neuronal Cell ◽

Rat Hepatocytes ◽

Cell Types ◽

Liver Cells ◽

Human Platelets ◽

Rat Liver Cells

The monohydroxy bile acid taurolithocholate permeabilizes the endoplasmic reticulum to Ca2+ in rat liver cells. To assess whether this action on the endoplasmic reticulum was restricted to this tissue, the effects of bile acid were investigated in two cell types quite unrelated to rat hepatocyte, namely human platelets and neuronal NG108-15 cell line. The results showed that taurolithocholate (3-100 microM) had no effect on free cytosolic [Ca2+] in human platelets and NG108-15 cells. whereas it increased it from 180 to 520 nM in rat hepatocytes. In contrast, in cells permeabilized by saponin, taurolithocholate initiated a profound release of the stored Ca2+ from the internal Ca2+ pools in the three cell types. The bile acid released 90% of the Ca2+ pools, with rate constants of about 5 min-1 and half-maximal effects at 15-30 microM. The results also showed that, in contrast with liver cells, which displayed an influx of [14C]taurolithocholate of 2 nmol/min per mg, human platelets and the neuronal cell line appeared to be resistant to [14C]taurolithocholate uptake. The influx measured in these latter cells was about 100-fold lower than in rat liver cells. Taken together, these data suggest that human platelets and NG108-15 cells do not possess the transport system for concentrating monohydroxy bile acids into cells. However, they show that human platelets and neuronal NG108-15 possess, in common with liver cells, the intracellular system responsible for taurolithocholate-mediated Ca2+ release from internal stores.

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Glucose regulates AMP-activated protein kinase activity and gene expression in clonal, hypothalamic neurons expressing proopiomelanocortin: additive effects of leptin or insulin

Journal of Endocrinology ◽

10.1677/joe-06-0080 ◽

2007 ◽

Vol 192 (3) ◽

pp. 605-614 ◽

Cited By ~ 46

Author(s):

Fang Cai ◽

Armen V Gyulkhandanyan ◽

Michael B Wheeler ◽

Denise D Belsham

Keyword(s):

Protein Kinase ◽

Energy Homeostasis ◽

Cell Model ◽

Neuronal Cell ◽

Cell Types ◽

Glucose Sensing ◽

Protein Kinase Activity ◽

Energy Status ◽

Ampk Activity ◽

Amp Activated Protein Kinase

The mammalian hypothalamus comprises an array of phenotypically distinct cell types that interpret peripheral signals of energy status and, in turn, elicits an appropriate response to maintain energy homeostasis. We used a clonal representative hypothalamic cell model expressing proopiomelanocortin (POMC; N-43/5) to study changes in AMP-activated protein kinase (AMPK) activity and glucose responsiveness. We have demonstrated the presence of cellular machinery responsible for glucose sensing in the cell line, including glucokinase, glucose transporters, and appropriate ion channels. ATP-sensitive potassium channels were functional and responded to glucose. The N-43/5 POMC neurons may therefore be an appropriate cell model to study glucose-sensing mechanisms in the hypothalamus. In N-43/5 POMC neurons, increasing glucose concentrations decreased phospho-AMPK activity. As a relevant downstream effect, we found that POMC transcription increased with 2.8 and 16.7 mM glucose. Upon addition of leptin, with either no glucose or with 5 mM glucose, we found that leptin decreased AMPK activity in N-43/5 POMC neurons, but had no significant effect at 25 mM glucose, whereas insulin decreased AMPK activity at only 5 mM glucose. These results demonstrate that individual hypothalamic neuronal cell types, such as the POMC neuron, can have distinct responses to peripheral signals that relay energy status to the brain, and will therefore be activated uniquely to control neuroendocrine function.

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From blastocyst to gastrula: gene regulatory networks of embryonic stem cells and early mouse embryogenesis

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2013.0542 ◽

2014 ◽

Vol 369 (1657) ◽

pp. 20130542 ◽

Cited By ~ 18

Author(s):

David-Emlyn Parfitt ◽

Michael M. Shen

Keyword(s):

Stem Cells ◽

Gene Networks ◽

Regulatory Networks ◽

Embryonic Stem ◽

Cell Lineage ◽

Network Models ◽

Cell Types ◽

Mammalian Development ◽

A Genome

To date, many regulatory genes and signalling events coordinating mammalian development from blastocyst to gastrulation stages have been identified by mutational analyses and reverse-genetic approaches, typically on a gene-by-gene basis. More recent studies have applied bioinformatic approaches to generate regulatory network models of gene interactions on a genome-wide scale. Such models have provided insights into the gene networks regulating pluripotency in embryonic and epiblast stem cells, as well as cell-lineage determination in vivo . Here, we review how regulatory networks constructed for different stem cell types relate to corresponding networks in vivo and provide insights into understanding the molecular regulation of the blastocyst–gastrula transition.

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The effect of strontium ranelate on titanium particle-induced periprosthetic osteolysis regulated by WNT/β-catenin signaling in vivo and in vitro

Bioscience Reports ◽

10.1042/bsr20203003 ◽

2021 ◽

Vol 41 (1) ◽

Author(s):

Bolun Wang ◽

Haohui Guo ◽

Tianxiang Geng ◽

Kening Sun ◽

Liang Zhang ◽

...

Keyword(s):

Bone Resorption ◽

Aseptic Loosening ◽

Cell Line ◽

Conditioned Medium ◽

Strontium Ranelate ◽

Cell Model ◽

Periprosthetic Osteolysis ◽

A Cell

Abstract Aseptic loosening following periprosthetic osteolysis is the primary complication that limits the lifetime of total joint arthroplasty (TJA). The wear particles trigger a chronic inflammation response in the periprosthetic tissue and turn over the bone balance to bone resorption. The present study aimed to investigate the possible effect and mechanism of strontium ranelate (SR), a clinically safe drug for osteoporosis, on particle-induced periprosthetic osteolysis. Thirty-six female C57BL/6j mice underwent tibial Ti-nail implantation to establish an animal model of aseptic loosening. After 12 weeks, micro-CT results showed that strontium ranelate could inhibit periprosthetic bone resorption. In vitro, Ti particles were used to stimulate RAW264.7 cell line to collect conditioned medium, and co-culture MC3T3-E1 cell line with conditioned medium to establish a cell model of aseptic loosening. The results of alkaline phosphatase (ALP) detection, immunofluorescence, and flow cytometry demonstrated that strontium ranelate could regulate the expression of OPG/RANKL, promote differentiation and mineralization, and inhibit apoptosis in osteoblasts. Moreover, we revealed that SR’s exerted its therapeutic effect by down-regulating sclerostin, thereby activating the Wnt/β-catenin signal pathway. Therefore, this research suggests that strontium ranelate could be a potential drug for the prevention and treatment of particle-induced aseptic loosening post-TJA.

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Cell type resolved co-expression networks of core clock genes in brain development

10.1101/2020.12.30.424790 ◽

2021 ◽

Author(s):

Surbhi Sharma ◽

Asgar Hussain Ansari ◽

Soundhar Ramasamy

Keyword(s):

Circadian Clock ◽

Single Cell ◽

Radial Glia ◽

Clock Genes ◽

Neuronal Cell ◽

Cell Types ◽

Developing Brain ◽

Knock Out

AbstractThe circadian clock regulates vital cellular processes by adjusting the physiology of the organism to daily changes in the environment. Rhythmic transcription of core Clock Genes (CGs) and their targets regulate these processes at the cellular level. Circadian clock disruption has been observed in people with neurodegenerative disorders like Alzheimer’s and Parkinson’s. Also, ablation of CGs during development has been shown to affect neurogenesis in both in vivo and in vitro models. Previous studies on the function of CGs in the brain have used knock-out models of a few CGs. However, a complete catalog of CGs in different cell types of the developing brain is not available and it is also tedious to obtain. Recent advancements in single-cell RNA sequencing (scRNA-seq) has revealed novel cell types and elusive dynamic cell states of the developing brain. In this study by using publicly available single-cell transcriptome datasets we systematically explored CGs-coexpressing networks (CGs-CNs) during embryonic and adult neurogenesis. Our meta-analysis reveals CGs-CNs in human embryonic radial glia, neurons and also in lesser studied non-neuronal cell types of the developing brain.

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Single cell RNA sequencing of the Strongylocentrotus purpuratus larva reveals the blueprint of major cell types and nervous system of a non-chordate deuterostome

eLife ◽

10.7554/elife.70416 ◽

2021 ◽

Vol 10 ◽

Author(s):

Periklis Paganos ◽

Danila Voronov ◽

Jacob M Musser ◽

Detlev Arendt ◽

Maria Ina Arnone

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Sea Urchin ◽

Regulatory Networks ◽

Sea Urchins ◽

Neuronal Cell ◽

Cell Types ◽

Molecular Fingerprint ◽

Larval Body ◽

Single Cell Rna Sequencing

Identifying the molecular fingerprint of organismal cell types is key for understanding their function and evolution. Here, we use single cell RNA sequencing (scRNA-seq) to survey the cell types of the sea urchin early pluteus larva, representing an important developmental transition from non-feeding to feeding larva. We identify 21 distinct cell clusters, representing cells of the digestive, skeletal, immune, and nervous systems. Further subclustering of these reveal a highly detailed portrait of cell diversity across the larva, including the identification of neuronal cell types. We then validate important gene regulatory networks driving sea urchin development and reveal new domains of activity within the larval body. Focusing on neurons that co-express Pdx-1 and Brn1/2/4, we identify an unprecedented number of genes shared by this population of neurons in sea urchin and vertebrate endocrine pancreatic cells. Using differential expression results from Pdx-1 knockdown experiments, we show that Pdx1 is necessary for the acquisition of the neuronal identity of these cells. We hypothesize that a network similar to the one orchestrated by Pdx1 in the sea urchin neurons was active in an ancestral cell type and then inherited by neuronal and pancreatic developmental lineages in sea urchins and vertebrates.

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In vitro neuroprotective activities of two distinct probiotic consortia

Beneficial Microbes ◽

10.3920/bm2018.0105 ◽

2019 ◽

Vol 10 (4) ◽

pp. 437-447 ◽

Cited By ~ 2

Author(s):

D.R. Michael ◽

T.S. Davies ◽

K.E. Loxley ◽

M.D. Allen ◽

M.A. Good ◽

...

Keyword(s):

Cell Model ◽

Neuronal Cell ◽

In Vivo Studies ◽

Intact Cells ◽

Bacterial Consortia ◽

Differentiated Cells ◽

Genes Encoding ◽

Induced Apoptosis

Neurodegeneration has been linked to changes in the gut microbiota and this study compares the neuroprotective capability of two bacterial consortia, known as Lab4 and Lab4b, using the established SH-SY5Y neuronal cell model. Firstly, varying total antioxidant capacities (TAC) were identified in the intact cells from each consortia and their secreted metabolites, referred to as conditioned media (CM). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Crystal Violet (CV) assays of cell viability revealed that Lab4 CM and Lab4b CM could induce similar levels of proliferation in SH-SY5Y cells and, despite divergent TAC, possessed a comparable ability to protect undifferentiated and retinoic acid-differentiated cells from the cytotoxic actions of rotenone and undifferentiated cells from the cytotoxic actions of 1-methyl-4-phenylpyridinium iodide (MPP+). Lab4 CM and Lab4b CM also had the ability to attenuate rotenone-induced apoptosis and necrosis with Lab4b inducing the greater effect. Both consortia showed an analogous ability to attenuate intracellular reactive oxygen species accumulation in SH-SY5Y cells although the differential upregulation of genes encoding glutathione reductase and superoxide dismutase by Lab4 CM and Lab4b CM, respectively, implicates the involvement of consortia-specific antioxidative mechanisms of action. This study implicates Lab4 and Lab4b as potential neuroprotective agents and justifies their inclusion in further in vivo studies.

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