Developing a Sensitive Reporter System for Monitoring of Pancreatic and Duodenal Homeobox Gene 1 (Pdx1) and Neurogenin 3 (Ngn3) – Mediated Transdifferentiation from Human Hepatic Cells into Insulin-Producing Beta-Like Cells

2014 ◽  
Vol 989-994 ◽  
pp. 1003-1006
Author(s):  
Chao Xie ◽  
William Donelan ◽  
Shun Lu ◽  
Li Jun Yang
Keyword(s):  
Fusion Protein ◽  
Cellular Differentiation ◽  
Homeobox Gene ◽  
Cell Types ◽  
Cell Type ◽  
Reporter System ◽  
Neurogenin 3 ◽  
Hepatic Cells ◽  
Insulin Producing Cells ◽  
Vp16 Fusion

It is well known that cellular differentiation is not a terminal process. Transdifferentiation is the conversion of one differentiated cell type to another. There are many examples of induced transdifferentiation between cell types by expression of ectopic transcription factors. Here we show that combined lentiviral expression of Pdx1 or Pdx1-VP16 fusion protein and Ngn3 can direct the transdifferentiation of hepatic cells into insulin producing cells. We showed that the Pdx1 or Pdx1-VP16 fusion protein and Ngn3 together synergistically increased transactivation for the insulin gene. This provides a useful model to study the transdifferentiation process.

scholarly journals A nervous system-specific subnuclear organelle in Caenorhabditis elegans

Genetics ◽  
2021 ◽  
Vol 217 (1) ◽  
Author(s):  
Kenneth Pham ◽  
Neda Masoudi ◽  
Eduardo Leyva-Díaz ◽  
Oliver Hobert
Keyword(s):  
Nervous System ◽  
Caenorhabditis Elegans ◽  
Homeobox Gene ◽  
Cell Types ◽  
Nuclear Bodies ◽  
Loss Of Function ◽  
Cell Type ◽  
Cell Type Specificity ◽  
Splicing Speckles ◽  
Polycomb Bodies

Abstract We describe here phase-separated subnuclear organelles in the nematode Caenorhabditis elegans, which we term NUN (NUclear Nervous system-specific) bodies. Unlike other previously described subnuclear organelles, NUN bodies are highly cell type specific. In fully mature animals, 4–10 NUN bodies are observed exclusively in the nucleus of neuronal, glial and neuron-like cells, but not in other somatic cell types. Based on co-localization and genetic loss of function studies, NUN bodies are not related to other previously described subnuclear organelles, such as nucleoli, splicing speckles, paraspeckles, Polycomb bodies, promyelocytic leukemia bodies, gems, stress-induced nuclear bodies, or clastosomes. NUN bodies form immediately after cell cycle exit, before other signs of overt neuronal differentiation and are unaffected by the genetic elimination of transcription factors that control many other aspects of neuronal identity. In one unusual neuron class, the canal-associated neurons, NUN bodies remodel during larval development, and this remodeling depends on the Prd-type homeobox gene ceh-10. In conclusion, we have characterized here a novel subnuclear organelle whose cell type specificity poses the intriguing question of what biochemical process in the nucleus makes all nervous system-associated cells different from cells outside the nervous system.

scholarly journals Transcription activation of early human development suggests DUX4 as an embryonic regulator

2017 ◽  
Author(s):  
Virpi Töhönen ◽  
Shintaro Katayama ◽  
Liselotte Vesterlund ◽  
Mona Sheikhi ◽  
Liselotte Antonsson ◽  
...  
Keyword(s):  
Single Cells ◽  
Homeobox Gene ◽  
Cell Types ◽  
Cell Type ◽  
Cell Stage ◽  
Repeat Elements ◽  
Telomere Elongation ◽  
Distinct Cell ◽  
Cell Embryo ◽  
Gene Transcripts

In order to better understand human preimplantation development we applied massively parallel RNA sequencing on 337 single cells from oocytes up to 8-cell embryo blastomeres. Surprisingly, already before zygote pronuclear fusion we observed drastic changes in the transcriptome compared to the unfertilized egg: 1,804 gene transcripts and 32 repeat elements become more abundant, among these the double-homeobox geneDUX4. Several genes previously identified asDUX4targets, such asCCNA1, KHDC1LandZSCAN4, as well as several members of theRFPLs, TRIMSandPRAMEFs1were accumulated in 4-cell stage blastomeres, suggestingDUX4as an early regulator. In the 8-cell stage, we observed two distinct cell types – a transcript-poor cell type, and a transcript-rich cell type with many Alu repeats and accumulated markers for pluripotency and stemness, telomere elongation and growth. In summary, this unprecedented detailed view of the first three days of human embryonic development reveals more complex changes in the transcriptome than what was previously known.

scholarly journals Salmonella endorses a dormant state within human epithelial cells for persistent infection

2020 ◽  
Author(s):  
Chak Hon Luk ◽  
Yuen-Yan Chang ◽  
Jost Enninga
Keyword(s):  
Flow Cytometry ◽  
Epithelial Cells ◽  
Fluorescence Microscopy ◽  
Host Cell ◽  
Cell Types ◽  
Host Cells ◽  
Cell Type ◽  
Reporter System ◽  
Fluorescent Reporter ◽  
Wide Range

AbstractSalmonella Typhimurium (S. Typhimurium) is an enteric bacterium capable of invading a wide range of hosts, including rodents and humans. It targets different host cell types showing different intracellular lifestyles. Within the infected cells S. Typhimurium colonizes multiple intracellular niches, and it is able to either actively divide at various rates, or remain dormant to persist. A comprehensive tool to monitor these distinct S. Typhimurium lifestyles has not been available so far. Here we developed a novel fluorescent reporter, Salmonella Intracellular Analyzer (SINA), compatible for fluorescence microscopy and flow cytometry for quantification at the single-bacterium level. Using SINA, we identified a S. Typhimurium subpopulation in infected epithelial cells that exhibits a unique phenotype in comparison to the previously documented vacuolar or cytosolic S. Typhimurium. This newly identified subpopulation remained dormant within a vesicular compartment distinct from either conventional Salmonella-containing vacuoles (SCV) or the previously reported niche of dormant S. Typhimurium inside macrophages. The dormant S. Typhimurium inside enterocytes were viable and expressed Salmonella Pathogenicity Island 2 (SPI-2) virulence factors at later infection time points. We found that the formation of these dormant S. Typhimurium is not triggered by the loss of SPI-2 expression but it is regulated by (p)ppGpp-mediated stringent response through RelA and SpoT. We predict that intraepithelial dormant S. Typhimurium represents an important pathogen niche as it provides an alternative strategy for S. Typhimurium pathogenicity and persistence.Author SummarySalmonella Typhimurium is a clinically relevant bacterial pathogen that causes Salmonellosis. It can actively or passively invade various host cell types and reside in a Salmonella-containing vacuole (SCV) within host cells. The SCV can be remodeled into a replicative niche with the aid of Salmonella Type III Secretion System 2 (T3SS2) effectors or else, the SCV is ruptured for the access of the nutrient-rich host cytosol. Depending on the infected host cell type, S. Typhimurium undertake different lifestyles that are distinct by their subcellular localization, replication rate and metabolic rate. We present here a novel fluorescent reporter system that rapidly detects S. Typhimurium lifestyles using fluorescence microscopy and flow cytometry. We identified a dormant S. Typhimurium population within enterocytes that displays capacities in host cell persistence, dormancy exit and antibiotic tolerance. We found that the molecular pathway suppressing S. Typhimurium dormancy in enterocytes is the one that has been shown to promote dormancy in macrophages. This suggests a divergent physiological consequence regulated by the same set of S. Typhimurium molecular mediators depending on the challenged host cell type. Altogether, our work demonstrates the potential of fluorescence reporters in facile bacterial characterization, and revealed a dormant S. Typhimurium population in human enterocytes that is distinct from those observed in macrophages and fibroblasts.

The cloning cycle: from amphibia to mammals and back

2001 ◽  
Vol 9 (1) ◽  
pp. 3-31 ◽  
Author(s):  
IM Lewis ◽  
MJ Munsie ◽  
AJ French ◽  
R Daniels ◽  
AO Trounson
Keyword(s):  
Developmental Biology ◽  
Nuclear Transfer ◽  
Cellular Differentiation ◽  
Cell Types ◽  
Nuclear Material ◽  
Laboratory Animals ◽  
Cell Type ◽  
Differentiated Cells ◽  
Different Cell Types ◽  
Fertilized Egg

The process whereby a fertilized egg develops into more than 200 different cell types and forms a new individual is both intriguing and fundamental to developmental biology. The systematic pathway of cellular differentiation is generally thought to confer a restriction whereby differentiated cells lose the capacity to change into other cell types. However, the substitution of nuclear material from one cell type (egg) with that of another, in a process known as nuclear transfer or cloning, can reverse this restriction by removing epigenetic modifications to the chromatin structure. The ability to clone new individuals using this procedure was achieved some 30 years ago in amphibia in an effort to understand cellular differentiation. More recently, with the advent of improved embryo micromanipulation techniques, the technology has been applied to domestic and laboratory animals.

Dendritic cells of the human epidermis: immuno-electron microscopic studies of la antigen reactivity

1978 ◽  
Vol 36 (2) ◽  
pp. 644-645
Author(s):  
G. Rowden ◽  
M. G. Lewis ◽  
T. M. Phillips
Keyword(s):  
Dendritic Cells ◽  
Langerhans Cells ◽  
Cell Types ◽  
Cell Type ◽  
Electron Microscopic ◽  
La Antigen ◽  
Microscopic Studies ◽  
A Cell ◽  
C3 Receptors ◽  
Antigen Reactivity

Langerhans cells of mammalian stratified squamous epithelial have proven to be an enigma since their discovery in 1868. These dendritic suprabasal cells have been considered as related to melanocytes either as effete cells, or as post divisional products. Although grafting experiments seemed to demonstrate the independence of the cell types, much confusion still exists. The presence in the epidermis of a cell type with morphological features seemingly shared by melanocytes and Langerhans cells has been especially troublesome. This so called "indeterminate", or " -dendritic cell" lacks both Langerhans cells granules and melanosomes, yet it is clearly not a keratinocyte. Suggestions have been made that it is related to either Langerhans cells or melanocyte. Recent studies have unequivocally demonstrated that Langerhans cells are independent cells with immune function. They display Fc and C3 receptors on their surface as well as la (immune region associated) antigens.

scholarly journals Fibroblasts Influence the Efficacy, Resistance, and Future Use of Vaccines and Immunotherapy in Cancer Treatment

Vaccines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 634
Author(s):  
Bailee H. Sliker ◽  
Paul M. Campbell
Keyword(s):  
Transforming Growth Factor ◽  
Transforming Growth Factor Β ◽  
Cell Types ◽  
Fibroblast Activation Protein ◽  
Cancer Associated Fibroblasts ◽  
Cell Type ◽  
Adaptive Immune ◽  
Adaptive Immune Cells ◽  
New Research ◽  
Tumor Types

Tumors are composed of not only epithelial cells but also many other cell types that contribute to the tumor microenvironment (TME). Within this space, cancer-associated fibroblasts (CAFs) are a prominent cell type, and these cells are connected to an increase in tumor progression as well as alteration of the immune landscape present in and around the tumor. This is accomplished in part by their ability to alter the presence of both innate and adaptive immune cells as well as the release of various chemokines and cytokines, together leading to a more immunosuppressive TME. Furthermore, new research implicates CAFs as players in immunotherapy response in many different tumor types, typically by blunting their efficacy. Fibroblast activation protein (FAP) and transforming growth factor β (TGF-β), two major CAF proteins, are associated with the outcome of different immunotherapies and, additionally, have become new targets themselves for immune-based strategies directed at CAFs. This review will focus on CAFs and how they alter the immune landscape within tumors, how this affects response to current immunotherapy treatments, and how immune-based treatments are currently being harnessed to target the CAF population itself.

scholarly journals Epigenetic reprogramming of cell identity: lessons from development for regenerative medicine

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Amitava Basu ◽  
Vijay K. Tiwari
Keyword(s):  
Regenerative Medicine ◽  
Cell Types ◽  
Direct Conversion ◽  
Cellular Reprogramming ◽  
Specific Cell ◽  
Cell Type ◽  
Pathological Conditions ◽  
Gene Regulatory ◽  
Induced Pluripotent ◽  
And Function

AbstractEpigenetic mechanisms are known to define cell-type identity and function. Hence, reprogramming of one cell type into another essentially requires a rewiring of the underlying epigenome. Cellular reprogramming can convert somatic cells to induced pluripotent stem cells (iPSCs) that can be directed to differentiate to specific cell types. Trans-differentiation or direct reprogramming, on the other hand, involves the direct conversion of one cell type into another. In this review, we highlight how gene regulatory mechanisms identified to be critical for developmental processes were successfully used for cellular reprogramming of various cell types. We also discuss how the therapeutic use of the reprogrammed cells is beginning to revolutionize the field of regenerative medicine particularly in the repair and regeneration of damaged tissue and organs arising from pathological conditions or accidents. Lastly, we highlight some key challenges hindering the application of cellular reprogramming for therapeutic purposes.

scholarly journals Connectivity characterization of the mouse basolateral amygdalar complex

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Houri Hintiryan ◽  
Ian Bowman ◽  
David L. Johnson ◽  
Laura Korobkova ◽  
Muye Zhu ◽  
...  
Keyword(s):  
Granular Cell ◽  
Cell Types ◽  
Projection Neurons ◽  
Cell Type ◽  
Connectivity Map ◽  
Analysis Techniques ◽  
Domain Specific ◽  
Cell Type Specific ◽  
Unique Domain

AbstractThe basolateral amygdalar complex (BLA) is implicated in behaviors ranging from fear acquisition to addiction. Optogenetic methods have enabled the association of circuit-specific functions to uniquely connected BLA cell types. Thus, a systematic and detailed connectivity profile of BLA projection neurons to inform granular, cell type-specific interrogations is warranted. Here, we apply machine-learning based computational and informatics analysis techniques to the results of circuit-tracing experiments to create a foundational, comprehensive BLA connectivity map. The analyses identify three distinct domains within the anterior BLA (BLAa) that house target-specific projection neurons with distinguishable morphological features. We identify brain-wide targets of projection neurons in the three BLAa domains, as well as in the posterior BLA, ventral BLA, posterior basomedial, and lateral amygdalar nuclei. Inputs to each nucleus also are identified via retrograde tracing. The data suggests that connectionally unique, domain-specific BLAa neurons are associated with distinct behavior networks.

scholarly journals Shisa6 mediates cell-type specific regulation of depression in the nucleus accumbens

2021 ◽  
Author(s):  
Hee-Dae Kim ◽  
Jing Wei ◽  
Tanessa Call ◽  
Nicole Teru Quintus ◽  
Alexander J. Summers ◽  
...  
Keyword(s):  
Nucleus Accumbens ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Current Treatment ◽  
Specific Cell ◽  
Excitatory Synapses ◽  
Cell Type ◽  
Cell Type Specific ◽  
Specific Regulation ◽  
Circuit Function

AbstractDepression is the leading cause of disability and produces enormous health and economic burdens. Current treatment approaches for depression are largely ineffective and leave more than 50% of patients symptomatic, mainly because of non-selective and broad action of antidepressants. Thus, there is an urgent need to design and develop novel therapeutics to treat depression. Given the heterogeneity and complexity of the brain, identification of molecular mechanisms within specific cell-types responsible for producing depression-like behaviors will advance development of therapies. In the reward circuitry, the nucleus accumbens (NAc) is a key brain region of depression pathophysiology, possibly based on differential activity of D1- or D2- medium spiny neurons (MSNs). Here we report a circuit- and cell-type specific molecular target for depression, Shisa6, recently defined as an AMPAR component, which is increased only in D1-MSNs in the NAc of susceptible mice. Using the Ribotag approach, we dissected the transcriptional profile of D1- and D2-MSNs by RNA sequencing following a mouse model of depression, chronic social defeat stress (CSDS). Bioinformatic analyses identified cell-type specific genes that may contribute to the pathogenesis of depression, including Shisa6. We found selective optogenetic activation of the ventral tegmental area (VTA) to NAc circuit increases Shisa6 expression in D1-MSNs. Shisa6 is specifically located in excitatory synapses of D1-MSNs and increases excitability of neurons, which promotes anxiety- and depression-like behaviors in mice. Cell-type and circuit-specific action of Shisa6, which directly modulates excitatory synapses that convey aversive information, identifies the protein as a potential rapid-antidepressant target for aberrant circuit function in depression.

scholarly journals Histone modifications form a cell-type-specific chromosomal bar code that persists through the cell cycle

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
John A. Halsall ◽  
Simon Andrews ◽  
Felix Krueger ◽  
Charlotte E. Rutledge ◽  
Gabriella Ficz ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Histone Modifications ◽  
Expression Patterns ◽  
Cell Types ◽  
Cell Type ◽  
Bar Code ◽  
Genes Encoding ◽  
Cell Type Specific ◽  
Rolling Windows

AbstractChromatin configuration influences gene expression in eukaryotes at multiple levels, from individual nucleosomes to chromatin domains several Mb long. Post-translational modifications (PTM) of core histones seem to be involved in chromatin structural transitions, but how remains unclear. To explore this, we used ChIP-seq and two cell types, HeLa and lymphoblastoid (LCL), to define how changes in chromatin packaging through the cell cycle influence the distributions of three transcription-associated histone modifications, H3K9ac, H3K4me3 and H3K27me3. We show that chromosome regions (bands) of 10–50 Mb, detectable by immunofluorescence microscopy of metaphase (M) chromosomes, are also present in G1 and G2. They comprise 1–5 Mb sub-bands that differ between HeLa and LCL but remain consistent through the cell cycle. The same sub-bands are defined by H3K9ac and H3K4me3, while H3K27me3 spreads more widely. We found little change between cell cycle phases, whether compared by 5 Kb rolling windows or when analysis was restricted to functional elements such as transcription start sites and topologically associating domains. Only a small number of genes showed cell-cycle related changes: at genes encoding proteins involved in mitosis, H3K9 became highly acetylated in G2M, possibly because of ongoing transcription. In conclusion, modified histone isoforms H3K9ac, H3K4me3 and H3K27me3 exhibit a characteristic genomic distribution at resolutions of 1 Mb and below that differs between HeLa and lymphoblastoid cells but remains remarkably consistent through the cell cycle. We suggest that this cell-type-specific chromosomal bar-code is part of a homeostatic mechanism by which cells retain their characteristic gene expression patterns, and hence their identity, through multiple mitoses.

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