scholarly journals Paediatric gastric organoids as a tool for disease modelling and clinical translation

2021 ◽  
Vol 37 (3) ◽  
pp. 317-324
Author(s):  
Brendan C. Jones ◽  
Giuseppe Calà ◽  
Paolo De Coppi ◽  
Giovanni Giuseppe Giobbe
Keyword(s):  
Epithelial Cell ◽  
Single Cell ◽  
3D Culture ◽  
Cell Types ◽  
Defined Medium ◽  
Gastric Epithelium ◽  
Apical Surface ◽  
Gastric Glands ◽  
Cell Dissociation

Abstract Purpose Knowledge of gastric epithelial homeostasis remains incomplete, lacking human-specific models for study. This study establishes a protocol for deriving gastric epithelial organoids from paediatric gastric biopsies, providing a platform for modelling disease and developing translational therapies. Methods Full-thickness surgical samples and endoscopic mucosal biopsies were obtained from six patients. Gastric glands were isolated by a chemical chelation protocol and then plated in 3D culture in Matrigel® droplets in chemically defined medium. After formation, organoids were passaged by single cell dissociation or manual disaggregation. Cell composition and epithelial polarity of organoids were assessed by bright field microscopy and immunofluorescence analysis, comparing them to native paediatric gastric tissue. Results Gastric glands were successfully isolated from all six patients who were aged 4 months to 16 years. Gastric glands from all patients sealed to form spherical gastric organoids. These organoids could be passaged by manual disaggregation or single cell dissociation, remaining proliferative up to 1 year in culture. Organoids retained normal epithelial cell polarity, with the apical surface orientated towards the central lumen. Organoids expressed markers of mature gastric epithelial cell types, except for parietal cells. Conclusion Gastric organoids can be reliably generated from paediatric biopsies and are a representative in vitro model for studying gastric epithelium.

Selection of kidney cell types in primary glomerular explant outgrowths by in vitro culture conditions

1986 ◽  
Vol 84 (1) ◽  
pp. 69-92
Author(s):  
T.D. Oberley ◽  
A.H. Yang ◽  
J. Gould-Kostka
Keyword(s):  
Epithelial Cell ◽  
Cell Types ◽  
Defined Medium ◽  
Culture Conditions ◽  
Chemically Defined Medium ◽  
Cell Type ◽  
Epithelial Cell Type ◽  
Tubular Cells ◽  
Selection Of

Adult guinea pig glomeruli were grown in vitro either in serum or in a chemically defined medium. Glomeruli were plated either directly into plastic flasks or into plastic flasks that had been coated with the extracellular matrix produced by the PF-HR-9 mouse teratocarcinoma endodermal cell line. Both the composition of the medium and the nature of the culture substrate affected whole glomerular attachment and the type of cells produced in culture. Quantitative studies demonstrated selection of cell types by different culture conditions. Three colony types, each composed of distinctive cell types, could be identified by morphological features. The cells constituting two of these colony types were epithelial in nature, but they were identified as different epithelial types by both histochemical and ultrastructural criteria. Previous studies suggested that one epithelial cell type was derived from the glomerular visceral epithelial cell. This study demonstrates that this cell type could be selectively grown in defined medium on plastic. A second cell type showed several features of renal tubular epithelial cells, including histochemical staining for catalase, cell surface microvilli and cilia, and formation of hemicysts and structures that resembled tubules after prolonged periods in culture. To demonstrate that the ‘glomerulus-derived’ tubular cells were indeed tubular epithelium, we isolated purified renal cortical tubules (greater than 99% pure) and cultured them on the HR-9 matrix in a serum-free chemically defined medium. The resultant outgrowths had morphological properties identical to those of the glomerulus-derived tubular cells. It seems likely that small tubular fragments attached to a minority of the glomeruli are the source of these glomerulus-derived tubular cells. Neither epithelial cell type could be subcultured on plastic, but both could be passaged on the HR-9 matrix. A third cell type, the spindle-shaped cell, was easily propagated on both plastic and the HR-9 matrix. The origin of this cell type is not clear. Our results demonstrate the important effect of culture conditions on the selection, growth and differentiation of kidney cell types in vitro.

A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype

1990 ◽  
Vol 259 (6) ◽  
pp. L415-L425 ◽  
Author(s):  
P. E. Roberts ◽  
D. M. Phillips ◽  
J. P. Mather
Keyword(s):  
Epithelial Cell ◽  
Molecular Mechanisms ◽  
Basal Medium ◽  
Fold Increase ◽  
Cell Types ◽  
Cell Number ◽  
Neonatal Rat ◽  
Rat Lung ◽  
Cell Type

A novel epithelial cell from normal neonatal rat lung has been isolated, established, and maintained for multiple passages in the absence of serum, without undergoing crisis or senescence. By careful manipulation of the nutrition/hormonal microenvironment, we have been able to select, from a heterogeneous population, a single epithelial cell type that can maintain highly differentiated features in vitro. This cell type has characteristics of bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observed for greater than 2 yr in continuous culture. It has been characterized by ultrastructural, morphological, and biochemical criteria. The basal medium for this cell line is Ham's F12/Dulbecco's modified Eagle's (DME) medium plus insulin (1 micrograms/ml), human transferrin (10 micrograms/ml), ethanolamine (10(-4) M), phosphoethanolamine (10(-4) M), selenium (2.5 x 10(-8) M), hydrocortisone (2.5 x 10(-7) M), and forskolin (5 microM). The addition of 150 micrograms/ml of bovine pituitary extract to the defined basal medium stimulates a greater than 10-fold increase in cell number and a 50- to 100-fold increase in thymidine incorporation. The addition of retinoic acid results in further enhancement of cell growth and complete inhibition of keratinization. We have demonstrated a strategy that may be applicable to isolating other cell types from the lung and maintaining their differentiated characteristics for long-term culture in vitro. Such a culture system promises to be a useful model in which to study cellular events associated with differentiation and proliferation in the lung and to better understand the molecular mechanisms involved in these events.

scholarly journals Single-cell analysis revealed that IL4I1 promoted ovarian cancer progression

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Hongyu Zhao ◽  
Yu Teng ◽  
Wende Hao ◽  
Jie Li ◽  
Zhefeng Li ◽  
...  
Keyword(s):  
Ovarian Cancer ◽  
Single Cell ◽  
Cancer Progression ◽  
Cox Regression ◽  
Single Cell Analysis ◽  
Cell Types ◽  
In Vitro Assays ◽  
Migration And Invasion ◽  
Dominant Type

Abstract Background Ovarian cancer was one of the leading causes of female deaths. Patients with OC were essentially incurable and portends a poor prognosis, presumably because of profound genetic heterogeneity limiting reproducible prognostic classifications. Methods We comprehensively analyzed an ovarian cancer single-cell RNA sequencing dataset, GSE118828, and identified nine major cell types. Relationship between the clusters was explored with CellPhoneDB. A malignant epithelial cluster was confirmed using pseudotime analysis, CNV and GSVA. Furthermore, we constructed the prediction model (i.e., RiskScore) consisted of 10 prognosis-specific genes from 2397 malignant epithelial genes using the LASSO Cox regression algorithm based on public datasets. Then, the prognostic value of Riskscore was assessed with Kaplan–Meier survival analysis and time-dependent ROC curves. At last, a series of in-vitro assays were conducted to explore the roles of IL4I1, an important gene in Riskscore, in OC progression. Results We found that macrophages possessed the most interaction pairs with other clusters, and M2-like TAMs were the dominant type of macrophages. C0 was identified as the malignant epithelial cluster. Patients with a lower RiskScore had a greater OS (log-rank P < 0.01). In training set, the AUC of RiskScore was 0.666, 0.743 and 0.809 in 1-year, 3-year and 5-year survival, respectively. This was also validated in another two cohorts. Moreover, downregulation of IL4I1 inhibited OC cells proliferation, migration and invasion. Conclusions Our work provide novel insights into our understanding of the heterogeneity among OCs, and would help elucidate the biology of OC and provide clinical guidance in prognosis for OC patients.

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

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.

scholarly journals Enhancing droplet-based single-nucleus RNA-seq resolution using the semi-supervised machine learning classifier DIEM

2019 ◽  
Author(s):  
Marcus Alvarez ◽  
Elior Rahmani ◽  
Brandon Jew ◽  
Kristina M. Garske ◽  
Zong Miao ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Cell Types ◽  
Supervised Machine Learning ◽  
Data Sets ◽  
Rna Seq ◽  
Novel Approach ◽  
Single Nucleus ◽  
Downstream Analysis

AbstractSingle-nucleus RNA sequencing (snRNA-seq) measures gene expression in individual nuclei instead of cells, allowing for unbiased cell type characterization in solid tissues. Contrary to single-cell RNA seq (scRNA-seq), we observe that snRNA-seq is commonly subject to contamination by high amounts of extranuclear background RNA, which can lead to identification of spurious cell types in downstream clustering analyses if overlooked. We present a novel approach to remove debris-contaminated droplets in snRNA-seq experiments, called Debris Identification using Expectation Maximization (DIEM). Our likelihood-based approach models the gene expression distribution of debris and cell types, which are estimated using EM. We evaluated DIEM using three snRNA-seq data sets: 1) human differentiating preadipocytes in vitro, 2) fresh mouse brain tissue, and 3) human frozen adipose tissue (AT) from six individuals. All three data sets showed various degrees of extranuclear RNA contamination. We observed that existing methods fail to account for contaminated droplets and led to spurious cell types. When compared to filtering using these state of the art methods, DIEM better removed droplets containing high levels of extranuclear RNA and led to higher quality clusters. Although DIEM was designed for snRNA-seq data, we also successfully applied DIEM to single-cell data. To conclude, our novel method DIEM removes debris-contaminated droplets from single-cell-based data fast and effectively, leading to cleaner downstream analysis. Our code is freely available for use at https://github.com/marcalva/diem.

scholarly journals Single Cell, Single Nucleus and Spatial RNA Sequencing of the Human Liver Identifies Hepatic Stellate Cell and Cholangiocyte Heterogeneity

2021 ◽  
Author(s):  
Tallulah S Andrews ◽  
Jawairia Atif ◽  
Jeff C Liu ◽  
Catia T Perciani ◽  
Xue-Zhong Ma ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Human Liver ◽  
Stellate Cell ◽  
Parenchymal Cell ◽  
Cell Types ◽  
Cell Populations ◽  
Healthy Human ◽  
Single Nucleus

The critical functions of the human liver are coordinated through the interactions of hepatic parenchymal and non-parenchymal cells. Recent advances in single cell transcriptional approaches have enabled an examination of the human liver with unprecedented resolution. However, dissociation related cell perturbation can limit the ability to fully capture the human liver's parenchymal cell fraction, which limits the ability to comprehensively profile this organ. Here, we report the transcriptional landscape of 73,295 cells from the human liver using matched single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq). The addition of snRNA-seq enabled the characterization of interzonal hepatocytes at single-cell resolution, revealed the presence of rare subtypes of hepatic stellate cells previously only seen in disease, and detection of cholangiocyte progenitors that had only been observed during in vitro differentiation experiments. However, T and B lymphocytes and NK cells were only distinguishable using scRNA-seq, highlighting the importance of applying both technologies to obtain a complete map of tissue-resident cell-types. We validated the distinct spatial distribution of the hepatocyte, cholangiocyte and stellate cell populations by an independent spatial transcriptomics dataset and immunohistochemistry. Our study provides a systematic comparison of the transcriptomes captured by scRNA-seq and snRNA-seq and delivers a high-resolution map of the parenchymal cell populations in the healthy human liver.

scholarly journals CellMap: Characterizing the types and composition of iPSC-derived cells from RNA-seq data

2021 ◽  
Author(s):  
Zhengyu Ouyang ◽  
Nathanael Bourgeois ◽  
Eugenia Lyashenko ◽  
Paige Cundiff ◽  
Patrick F Cullen ◽  
...  
Keyword(s):  
Single Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Types ◽  
Model Systems ◽  
Rna Seq ◽  
Cell Type ◽  
Fine Grained ◽  
Single Nucleus ◽  
Induced Pluripotent

Induced pluripotent stem cell (iPSC) derived cell types are increasingly employed as in vitro model systems for drug discovery. For these studies to be meaningful, it is important to understand the reproducibility of the iPSC-derived cultures and their similarity to equivalent endogenous cell types. Single-cell and single-nucleus RNA sequencing (RNA-seq) are useful to gain such understanding, but they are expensive and time consuming, while bulk RNA-seq data can be generated quicker and at lower cost. In silico cell type decomposition is an efficient, inexpensive, and convenient alternative that can leverage bulk RNA-seq to derive more fine-grained information about these cultures. We developed CellMap, a computational tool that derives cell type profiles from publicly available single-cell and single-nucleus datasets to infer cell types in bulk RNA-seq data from iPSC-derived cell lines.

Isolation and Culture of Single Cell Types from Rat Liver

2016 ◽  
Vol 201 (4) ◽  
pp. 253-267 ◽  
Author(s):  
Qidi Zhang ◽  
Ying Qu ◽  
Zhenghong Li ◽  
Qingqing Zhang ◽  
Mingyi Xu ◽  
...  
Keyword(s):  
Single Cell ◽  
Rat Liver ◽  
Gradient Centrifugation ◽  
Cell Types ◽  
Ethylene Glycol Tetraacetic Acid ◽  
High Yield ◽  
Isolated Cells ◽  
Liver Sinusoidal Endothelial Cells ◽  
Pathology Of The Liver

There have been few reports on the simultaneous isolation of multiple liver cell populations thus far. As such, this study was aimed at establishing a protocol for the simultaneous separation of hepatocytes (HCs), hepatic stellate cells (HSCs), liver sinusoidal endothelial cells (LSECs) and Kupffer cells (KCs) from the rat liver and assessing the in vitro culture of these cells. Single-cell suspensions from the liver were obtained by ethylene glycol tetraacetic acid/collagenase perfusion. After low-speed centrifugal separation of HCs, pronase was added to the nonparenchymal cell fraction to eliminate the remaining HCs. Subsequently, HSCs, LSECs and KCs were purified by two steps of density gradient centrifugation using Nycodenz and Percoll in addition to selective attachment. Pronase treatment increased the HSC yield (1.5 ± 0.2 vs. 0.7 ± 0.3 cells/g liver, p < 0.05) and improved LSEC purity (93.6 ± 3.6 vs. 82.5 ± 5.6%, p < 0.01). The isolated cells could also be cultured in vitro. LSEC apoptosis began on day 3 and reached a maximum on day 7. A few surviving LSECs began proliferating and split to form a cobblestone, sheet-like appearance on day 14. The LSECs on day 14 lost fenestrations but retained scavenger function. Thus, viable and purified liver cells were obtained with a high yield from the rat liver using the developed method, which may be useful for studying the physiology and pathology of the liver in the future.

scholarly journals Kidney organoid reproducibility across multiple human iPSC lines and diminished off target cells after transplantation revealed by single cell transcriptomics

2019 ◽  
Author(s):  
Ayshwarya Subramanian ◽  
Eriene-Heidi Sidhom ◽  
Maheswarareddy Emani ◽  
Nareh Sahakian ◽  
Katherine Vernon ◽  
...  
Keyword(s):  
Single Cell ◽  
Human Kidney ◽  
Cell Types ◽  
Mouse Kidney ◽  
Target Cells ◽  
Rna Seq ◽  
Kidney Capsule ◽  
Human Ipsc ◽  
Kidney Organoids

AbstractHuman iPSC-derived kidney organoids have the potential to revolutionize discovery, but assessing their consistency and reproducibility across iPSC lines, and reducing the generation of off-target cells remain an open challenge. Here, we used single cell RNA-Seq (scRNA-Seq) to profile 415,775 cells to show that organoid composition and development are comparable to human fetal and adult kidneys. Although cell classes were largely reproducible across iPSC lines, time points, protocols, and replicates, cell proportions were variable between different iPSC lines. Off-target cell proportions were the most variable. Prolonged in vitro culture did not alter cell types, but organoid transplantation under the mouse kidney capsule diminished off-target cells. Our work shows how scRNA-seq can help score organoids for reproducibility, faithfulness and quality, that kidney organoids derived from different iPSC lines are comparable surrogates for human kidney, and that transplantation enhances their formation by diminishing off-target cells.

scholarly journals Single-cell RNA-Seq Reveals Transcriptional Landscape and Intra-tumor Heterogenicity in Gallbladder Cancer Liver Metastasis Microenvironment

2020 ◽  
Author(s):  
Wenhua You ◽  
Xiangyu Li ◽  
Peng Wang ◽  
Bowen Sha ◽  
Yuan Liang ◽  
...  
Keyword(s):  
T Cells ◽  
Liver Metastasis ◽  
Gallbladder Cancer ◽  
Single Cell ◽  
Molecular Mechanisms ◽  
Cell Types ◽  
Malignant Cells ◽  
Different Cell Types ◽  
High Degree

Abstract Background: Gallbladder cancer (GBC) is a highly aggressive biliary epithelial malignancy. Tumor invasion and metastasis contributed to the high mortality of GBC patients. However, molecular mechanisms involved in GBC metastases are still little known. Methods: We performed single-cell RNA sequencing on GBC liver metastasis tissue and analyzed the data based on different cell types.Results: In this study, 8 cell types, including T cells, B cells, malignant cells, fibroblasts, endothelial cells, macrophages, dendritic cells, and mast cells were identified. Malignant cells displayed a high degree of intra-tumor heterogenicity and neutrophils could promote GBC progression in vitro. Besides, cytotoxic CD8+ T cells became exhausted and CD4+ Tregs presented immunosuppressive characteristics. Macrophages played an important role in the tumor microenvironment. We identified three distinct macrophage subsets and emerged M2 polarization. We also found that cancer-associated fibroblasts exhibited heterogeneity and promoted GBC metastasis. Conclusions: In conclusion, our work provided a landscape view at the single-cell level and may clear the way for the therapy of GBC metastases.

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