scholarly journals Cytokinesis and postabscission midbody remnants are regulated during mammalian brain development

2020 ◽  
Vol 117 (17) ◽  
pp. 9584-9593 ◽  
Author(s):  
Katrina C. McNeely ◽  
Noelle D. Dwyer
Keyword(s):  
Early Stage ◽  
Cell Types ◽  
Mammalian Brain ◽  
Daughter Cells ◽  
Proper Size ◽  
Steady Rate ◽  
Epithelial Structure ◽  
Live Image ◽  
Apical Membranes

Building a brain of the proper size and structure requires neural stem cells (NSCs) to divide with tight temporal and spatial control to produce different daughter cell types in proper numbers and sequence. Mammalian NSCs in the embryonic cortex must maintain their polarized epithelial structure as they undergo both early proliferative divisions and later neurogenic divisions. To do this, they undergo a polarized form of cytokinesis at the apical membrane that is not well understood. Here, we investigate whether polarized furrowing and abscission in mouse NSCs are regulated differently at earlier and later stages and in a cytokinesis mutant, Kif20b. This mutant was previously shown to have microcephaly and elevated apoptosis of NSCs. We developed methods to live image furrow ingression and midbody abscission in NSCs within cortical explants. We find that polarized furrow ingression occurs at a steady rate and completes in ∼15 min at two different ages. However, ingression is slower in a subset of Kif20b mutant NSCs. Abscission is usually observed on both sides of the midbody and takes 65 to 75 min to complete. Surprisingly, abscission is accelerated in the Kif20b mutant NSCs. Postabscission midbody remnants are observed at the apical membranes of daughter cells and are much more abundant in early-stage cortices. After NSC divisions in vitro, midbody remnants are more often retained on the daughter cells of early proliferative divisions. Altogether, these results suggest that regulation of abscission timing and midbody remnants in embryonic NSCs may influence proper brain growth and structure.

Recommended Guidelines for Developing, Qualifying, and Implementing Complex In Vitro Models (CIVMs) for Drug Discovery

2020 ◽  
Vol 25 (10) ◽  
pp. 1174-1190
Author(s):  
Jason E. Ekert ◽  
Julianna Deakyne ◽  
Philippa Pribul-Allen ◽  
Rebecca Terry ◽  
Christopher Schofield ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Early Stage ◽  
Cell Types ◽  
In Vitro Models ◽  
Success Rates ◽  
Lead Discovery ◽  
Cellular Models ◽  
Discovery Phase ◽  
Early Drug

The pharmaceutical industry is continuing to face high research and development (R&D) costs and low overall success rates of clinical compounds during drug development. There is an increasing demand for development and validation of healthy or disease-relevant and physiological human cellular models that can be implemented in early-stage discovery, thereby shifting attrition of future therapeutics to a point in discovery at which the costs are significantly lower. There needs to be a paradigm shift in the early drug discovery phase (which is lengthy and costly), away from simplistic cellular models that show an inability to effectively and efficiently reproduce healthy or human disease-relevant states to steer target and compound selection for safety, pharmacology, and efficacy questions. This perspective article covers the various stages of early drug discovery from target identification (ID) and validation to the hit/lead discovery phase, lead optimization, and preclinical safety. We outline key aspects that should be considered when developing, qualifying, and implementing complex in vitro models (CIVMs) during these phases, because criteria such as cell types (e.g., cell lines, primary cells, stem cells, and tissue), platform (e.g., spheroids, scaffolds or hydrogels, organoids, microphysiological systems, and bioprinting), throughput, automation, and single and multiplexing endpoints will vary. The article emphasizes the need to adequately qualify these CIVMs such that they are suitable for various applications (e.g., context of use) of drug discovery and translational research. The article ends looking to the future, in which there is an increase in combining computational modeling, artificial intelligence and machine learning (AI/ML), and CIVMs.

scholarly journals Fibroblast membrane-camouflaged nanoparticles for inflammation treatment in the early stage

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Lizhong Sun ◽  
Libang He ◽  
Wei Wu ◽  
Li Luo ◽  
Mingyue Han ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Immune Cell ◽  
Early Stage ◽  
Specific Treatment ◽  
Cell Types ◽  
Loss Of Function ◽  
Tissue Repair And Regeneration ◽  
Inflammatory Conditions

AbstractUnrestrained inflammation is harmful to tissue repair and regeneration. Immune cell membrane-camouflaged nanoparticles have been proven to show promise as inflammation targets and multitargeted inflammation controls in the treatment of severe inflammation. Prevention and early intervention of inflammation can reduce the risk of irreversible tissue damage and loss of function, but no cell membrane-camouflaged nanotechnology has been reported to achieve stage-specific treatment in these conditions. In this study, we investigated the prophylactic and therapeutic efficacy of fibroblast membrane-camouflaged nanoparticles for topical treatment of early inflammation (early pulpitis as the model) with the help of in-depth bioinformatics and molecular biology investigations in vitro and in vivo. Nanoparticles have been proven to act as sentinels to detect and competitively neutralize invasive Escherichia coli lipopolysaccharide (E. coli LPS) with resident fibroblasts to effectively inhibit the activation of intricate signaling pathways. Moreover, nanoparticles can alleviate the secretion of multiple inflammatory cytokines to achieve multitargeted anti-inflammatory effects, attenuating inflammatory conditions in the early stage. Our work verified the feasibility of fibroblast membrane-camouflaged nanoparticles for inflammation treatment in the early stage, which widens the potential cell types for inflammation regulation.

Identification of Adeno-Associated Virus Variants for Gene Transfer Into Human Neural Cell Types by Parallel Capsid Screening

2021 ◽  
Author(s):  
Lea Jessica Flitsch ◽  
Kathleen Börner ◽  
Christian Stüllein ◽  
Simon Ziegler ◽  
Vera Sonntag-Buck ◽  
...  
Keyword(s):  
Radial Glia ◽  
Disease Modeling ◽  
Early Stage ◽  
Cell Types ◽  
Neural Cell ◽  
Cell Type Specificity ◽  
Distinct Cell Type ◽  
Citrate Transporter ◽  
Human Neural Cell

Abstract Human brain cells generated by in vitrocell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant Adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, weharnessed a customizable parallel screening approach to assessa panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types.We identified common lead candidates suited for the transduction of directly converted,early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neuralprogenitors,as well as differentiated astrocytic and mixed neuroglial cultures.We then selected a subsetof these candidates for functional validation in iNSCs and iPSC-derived astrocytes, usingshRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview ofthe susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussionof the assets and limitations of the specific AAV capsidscreening approach.

scholarly journals Cross-species Transcriptomic Comparison of In Vitro and In Vivo Mammalian Neural Cells

2015 ◽  
Vol 9 ◽  
pp. BBI.S33124 ◽  
Author(s):  
Peter R. LoVerso ◽  
Christopher M. Wachter ◽  
Feng Cui
Keyword(s):  
Gene Expression ◽  
Transcript Abundance ◽  
Cell Types ◽  
Mammalian Brain ◽  
Neural Cells ◽  
Rna Seq ◽  
Commercial Sources ◽  
And Function

The mammalian brain is characterized by distinct classes of cells that differ in morphology, structure, signaling, and function. Dysregulation of gene expression in these cell populations leads to various neurological disorders. Neural cells often need to be acutely purified from animal brains for research, which requires complicated procedure and specific expertise. Primary culture of these cells in vitro is a viable alternative, but the differences in gene expression of cells grown in vitro and in vivo remain unclear. Here, we cultured three major neural cell classes of rat brain (ie, neurons, astrocytes, and oligodendrocyte precursor cells [OPCs]) obtained from commercial sources. We measured transcript abundance of these cell types by RNA sequencing (RNA-seq) and compared with their counterparts acutely purified from mouse brains. Cross-species RNA-seq data analysis revealed hundreds of genes that are differentially expressed between the cultured and acutely purified cells. Astrocytes have more such genes compared to neurons and OPCs, indicating that signaling pathways are greatly perturbed in cultured astrocytes. This dataset provides a powerful resource to demonstrate the similarities and differences of biological processes in mammalian neural cells grown in vitro and in vivo at the molecular level.

scholarly journals Strontium Promotes the Proliferation and Osteogenic Differentiation of Human Placental Decidual Basalis- and Bone Marrow-Derived MSCs in a Dose-Dependent Manner

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Yi-Zhou Huang ◽  
Cheng-Guang Wu ◽  
Hui-Qi Xie ◽  
Zhao-Yang Li ◽  
Antonietta Silini ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Early Stage ◽  
Cell Types ◽  
Osteogenic Potential ◽  
Dependent Manner ◽  
Gene Expressions ◽  
Dose Dependent ◽  
Osteogenic Gene

The osteogenic potential of mesenchymal stromal cells (MSCs) varies among different tissue sources. Strontium enhances the osteogenic differentiation of bone marrow-derived MSCs (BM-MSCs), but whether it exerts similar effects on placental decidual basalis-derived MSCs (PDB-MSCs) remains unknown. Here, we compared the influence of strontium on the proliferation and osteogenic differentiation of human PDB- and BM-MSCs in vitro. We found that 1 mM and 10 mM strontium, but not 0.1 mM strontium, evidently promoted the proliferation of human PDB- and BM-MSCs. These doses of strontium showed a comparable alkaline phosphatase activity in both cell types, but their osteogenic gene expressions were promoted in a dose-dependent manner. Strontium at doses of 0.1 mM and 1 mM elevated several osteogenic gene expressions of PDB-MSCs, but not those of BM-MSCs at an early stage. Nevertheless, they failed to enhance the mineralization of either cell type. By contrast, 10 mM strontium facilitated the osteogenic gene expression as well as the mineralization of human PDB- and BM-MSCs. Collectively, this study demonstrated that human PDB- and BM-MSCs shared a great similarity in response to strontium, which promoted their proliferation and osteogenic differentiation in a dose-dependent manner.

Germline and Somatic Mutations in the Hyaluronan Synthase–1 (HAS1) Gene May Contribute to Oncogenesis in Multiple Myeloma (MM) and Waldenstrom’s Macroglobulinemia (WM).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2488-2488
Author(s):  
Sophia Adamia ◽  
Amanda Reichert ◽  
Anirban Ghosh ◽  
Jennifer Hodges ◽  
Patrick Pilarski ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Plasma Cells ◽  
Mutational Analysis ◽  
Splice Variants ◽  
Early Stage ◽  
Cell Types ◽  
Host Genotype ◽  
Aberrant Splicing ◽  
The Impact

Abstract In MM and WM, we identified aberrant HAS1 splice variants that were absent from normal donors (HD) and B-CLL. Here we sequenced multiple subclones from multiple cell subsets to show that aberrant HAS1 splicing results from cryptic splice sites activation. Aberrant splicing defects are the consequences of genetic variations (GVs) detected in the sequence of classical splicing elements as well as within exons and introns. To investigate HAS1 splicing in MM and WM patients, we sequenced the HAS1 gene segments involved in abnormal splicing events. HAS1 from buccal epithelial cells (BEC) represented the host genotype, and hematopoietic progenitors (HP), T, B and plasma cells (PC) as the normal and malignant components of the hematopoietic lineage in MM/WM. 197 GV were found in 16 MM or WM patients, but none in 9B-CLL, MGUS or HD. We found 60 germline (defined as present in BEC and hematopoietic cells) and 137 somatic GV (defined as GV found in HP, T, B and/or PC, but absent from BEC). These somatic GV include 97 tumor-specific GV found in MM and/or WM B and PC and 40 hematopoietic origin GV identified in HP, T, B and PC, but not in BEC. Some GV were recurrent, detected in more than one patient. Recurrent GV (24 in MM and 22 in WM) included both germline and somatic GVs, 6 tumor-specific, 6 hematopoietic and 14 germline origin GV, as well as 20 NCBI-SNPs. The distribution of GV indicated that some of the recurrent germline and somatic GV are restricted to MM, some are restricted to WM and some are shared by both MM and WM. None were found in B-CLL, MGUS or HD. The patterns of germline GV observed in MM and WM suggests that MM and WM patients, but not B-CLL, inherit recurrent germline GV that are necessary but not sufficient for progression to malignancy. Acquisition of recurrent, somatic HAS1 GV in HP further increases the risk of developing MM or WM. Transformation may become inevitable when tumor-specific recurrent GV are acquired. Interestingly, we detected increased homozygosity for the mutated allele of some germline GV in all cell types (PC, B, T, HPs and BECs) from MM and/or WM patients. These GV were detected in 75-90% of subclones analyzed. Mutational analysis of minigene constructs demonstrated a distribution of GV as clusters in the vicinity of HAS1splicing elements, allowing us to classify them as “splicing mutations”. This is supported by an in vitro splicing assay, which confirmed that a combination of germline and somatic GVs leads to aberrant HAS1 splicing (see abstract by Kriangkum et al., ASH 2007). Our study demonstrates that the impact of inherited and acquired GV on HAS1 gene splicing is manifested only in the context of accompanying tumor-specific HAS1 GV, that in combination give rise to the clinically significant aberrant splicing of HAS1. It also indicates that MM and WM are closely related at the genomic level, with the same recurrent somatic mutations independently arising in both diseases. Accumulation of somatically acquired HAS1 mutations in HP, in the context of inherited predispositions to MM and WM may represent a very early stage of pre-malignant development. Similar to leukemias, initiation events that contribute to MM and WM pathogenesis may arise from mutations which first accumulate during the non-malignant or pre-malignant stages of hematopoietic differentiation in progenitor cells.

scholarly journals Wide-field and two-photon imaging of brain activity with voltage- and calcium-sensitive dyes

2009 ◽  
Vol 364 (1529) ◽  
pp. 2453-2467 ◽  
Author(s):  
Ryota Homma ◽  
Bradley J. Baker ◽  
Lei Jin ◽  
Olga Garaschuk ◽  
Arthur Konnerth ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Brain Activity ◽  
Cell Types ◽  
Mammalian Brain ◽  
Wide Field ◽  
Voltage Sensitive Dye ◽  
Two Photon ◽  
Advantages And Disadvantages ◽  
Photon Imaging

This review presents three examples of using voltage- or calcium-sensitive dyes to image the activity of the brain. Our aim is to discuss the advantages and disadvantages of each method with particular reference to its application to the study of the brainstem. Two of the examples use wide-field (one-photon) imaging; the third uses two-photon scanning microscopy. Because the measurements have limited signal-to-noise ratio, the paper also discusses the methodological aspects that are critical for optimizing the signal. The three examples are the following. (i) An intracellularly injected voltage-sensitive dye was used to monitor membrane potential in the dendrites of neurons in in vitro preparations. These experiments were directed at understanding how individual neurons convert complex synaptic inputs into the output spike train. (ii) An extracellular, bath application of a voltage-sensitive dye was used to monitor population signals from different parts of the dorsal brainstem. We describe recordings made during respiratory activity. The population signals indicated four different regions with distinct activity correlated with inspiration. (iii) Calcium-sensitive dyes can be used to label many individual cells in the mammalian brain. This approach, combined with two-photon microscopy, made it possible to follow the spike activity in an in vitro brainstem preparation during fictive respiratory rhythms. The organic voltage- and ion-sensitive dyes used today indiscriminatively stain all of the cell types in the preparation. A major effort is underway to develop fluorescent protein sensors of activity for selectively staining individual cell types.

scholarly journals Single-cell transcriptome analysis identifies distinct cell types and intercellular niche signaling in a primary gastric organoid model

2017 ◽  
Author(s):  
Jiamin Chen ◽  
Billy T Lau ◽  
Noemi Andor ◽  
Sue M Grimes ◽  
Christine Handy ◽  
...  
Keyword(s):  
Early Stage ◽  
Critical Role ◽  
Tumor Development ◽  
Cell Types ◽  
Liquid Interface ◽  
Stem Cell Marker ◽  
Intercellular Signaling ◽  
Tissue Microenvironment ◽  
Air Liquid Interface

ABSTRACTThe diverse cellular milieu of the gastric tissue microenvironment plays a critical role in normal tissue homeostasis and tumor development. However, few cell culture model can recapitulate the tissue microenvironment and intercellular signalingin vitro. Here we applied an air-liquid interface method to culture primary gastric organoids that contains epithelium with endogenous stroma. To characterize the microenvironment and intercellular signaling in this model, we analyzed the transcriptomes of over 5,000 individual cells from primary gastric organoids cultured at different time points. We identified epithelial cells, fibroblasts and macrophages at the early stage of organoid formation, and revealed that macrophages were polarized towards wound healing and tumor promotion. The organoids maintained both epithelial and fibroblast lineages during the course of time, and a subset of cells in both lineages expressed the stem cell markerLgr5. We identified thatRspo3was specifically expressed in the fibroblast lineage, providing an endogenous source of the R-spondin to activate Wnt signaling. Our studies demonstrate that air-liquid-interface-derived organoids provide a novel platform to study intercellular signaling and immune responsein vitro.

scholarly journals Defining the variety of cell types in developing and adult human kidneys by single-cell RNA sequencing

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
A. Schumacher ◽  
M. B. Rookmaaker ◽  
J. A. Joles ◽  
R. Kramann ◽  
T. Q. Nguyen ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Renal Cell ◽  
Early Stage ◽  
Kidney Tissue ◽  
Cell Types ◽  
Intermediate Cell ◽  
Adult Human ◽  
Single Cell Rna Sequencing

AbstractThe kidney is among the most complex organs in terms of the variety of cell types. The cellular complexity of human kidneys is not fully unraveled and this challenge is further complicated by the existence of multiple progenitor pools and differentiation pathways. Researchers disagree on the variety of renal cell types due to a lack of research providing a comprehensive picture and the challenge to translate findings between species. To find an answer to the number of human renal cell types, we discuss research that used single-cell RNA sequencing on developing and adult human kidney tissue and compares these findings to the literature of the pre-single-cell RNA sequencing era. We find that these publications show major steps towards the discovery of novel cell types and intermediate cell stages as well as complex molecular signatures and lineage pathways throughout development. The variety of cell types remains variable in the single-cell literature, which is due to the limitations of the technique. Nevertheless, our analysis approaches an accumulated number of 41 identified cell populations of renal lineage and 32 of non-renal lineage in the adult kidney, and there is certainly much more to discover. There is still a need for a consensus on a variety of definitions and standards in single-cell RNA sequencing research, such as the definition of what is a cell type. Nevertheless, this early-stage research already proves to be of significant impact for both clinical and regenerative medicine, and shows potential to enhance the generation of sophisticated in vitro kidney tissue.

Ultrastructural Changes in Three Different Mammalian Cells Following Ultraviolet Laser Irradiation

1972 ◽  
Vol 30 ◽  
pp. 350-351
Author(s):  
K. Shankar Narayan ◽  
Kailash C. Gupta ◽  
Tohru Okigaki
Keyword(s):  
Ultraviolet Light ◽  
Mammalian Cells ◽  
Biological Effects ◽  
Survival Rates ◽  
Chinese Hamster ◽  
Cell Types ◽  
Differential Sensitivity ◽  
Ultrastructural Changes ◽  
Ultraviolet Laser

The biological effects of short-wave ultraviolet light has generally been described in terms of changes in cell growth or survival rates and production of chromosomal aberrations. Ultrastructural changes following exposure of cells to ultraviolet light, particularly at 265 nm, have not been reported.We have developed a means of irradiating populations of cells grown in vitro to a monochromatic ultraviolet laser beam at a wavelength of 265 nm based on the method of Johnson. The cell types studies were: i) WI-38, a human diploid fibroblast; ii) CMP, a human adenocarcinoma cell line; and iii) Don C-II, a Chinese hamster fibroblast cell strain. The cells were exposed either in situ or in suspension to the ultraviolet laser (UVL) beam. Irradiated cell populations were studied either "immediately" or following growth for 1-8 days after irradiation.Differential sensitivity, as measured by survival rates were observed in the three cell types studied. Pattern of ultrastructural changes were also different in the three cell types.

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