High-Content Image-Based Single-Cell Phenotypic Analysis for the Testicular Toxicity Prediction Induced by Bisphenol A and Its Analogs Bisphenol S, Bisphenol AF, and Tetrabromobisphenol A in a Three-Dimensional Testicular Cell Co-culture Model

Abstract Emerging data indicate that structural analogs of bisphenol A (BPA) such as bisphenol S (BPS), tetrabromobisphenol A (TBBPA), and bisphenol AF (BPAF) have been introduced into the market as substitutes for BPA. Our previous study compared in vitro testicular toxicity using murine C18-4 spermatogonial cells and found that BPAF and TBBPA exhibited higher spermatogonial toxicities as compared with BPA and BPS. Recently, we developed a novel in vitro three-dimensional (3D) testicular cell co-culture model, enabling the classification of reproductive toxic substances. In this study, we applied the testicular cell co-culture model and employed a high-content image (HCA)-based single-cell analysis to further compare the testicular toxicities of BPA and its analogs. We also developed a machine learning (ML)-based HCA pipeline to examine the complex phenotypic changes associated with testicular toxicities. We found dose- and time-dependent changes in a wide spectrum of adverse endpoints, including nuclear morphology, DNA synthesis, DNA damage, and cytoskeletal structure in a single-cell-based analysis. The co-cultured testicular cells were more sensitive than the C18 spermatogonial cells in response to BPA and its analogs. Unlike conventional population-averaged assays, single-cell-based assays not only showed the levels of the averaged population, but also revealed changes in the sub-population. Machine learning-based phenotypic analysis revealed that treatment of BPA and its analogs resulted in the loss of spatial cytoskeletal structure, and an accumulation of M phase cells in a dose- and time-dependent manner. Furthermore, treatment of BPAF-induced multinucleated cells, which were associated with altered DNA damage response and impaired cellular F-actin filaments. Overall, we demonstrated a new and effective means to evaluate multiple toxic endpoints in the testicular co-culture model through the combination of ML and high-content image-based single-cell analysis. This approach provided an in-depth analysis of the multi-dimensional HCA data and provided an unbiased quantitative analysis of the phenotypes of interest.

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Generation of three-dimensional pannus-like tissues in vitro from single cell suspensions of synovial fluid cells from arthritis patients

Rheumatology International ◽

10.1007/s00296-003-0333-0 ◽

2004 ◽

Vol 24 (2) ◽

pp. 71-76 ◽

Cited By ~ 1

Author(s):

Samuel Solomon ◽

Madhan Masilamani ◽

Subhasis Mohanty ◽

J�rg E. Schwab ◽

Eva-Maria Boneberg ◽

...

Keyword(s):

Synovial Fluid ◽

Single Cell ◽

Three Dimensional ◽

Cell Suspensions ◽

Synovial Fluid Cells

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Fabrication of high-aspect-ratio microstructures in polymer microfluid chips for in vitro single-cell analysis

Technical Physics ◽

10.1134/s106378421610008x ◽

2016 ◽

Vol 61 (10) ◽

pp. 1566-1571 ◽

Cited By ~ 10

Author(s):

A. S. Bukatin ◽

I. S. Mukhin ◽

E. I. Malyshev ◽

I. V. Kukhtevich ◽

A. A. Evstrapov ◽

...

Keyword(s):

Aspect Ratio ◽

Single Cell ◽

High Aspect Ratio ◽

Single Cell Analysis ◽

Cell Analysis

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Single Cell Analysis Reveals That IL-4 Receptor/Stat6 Signaling Is Not Required for the In Vivo or In Vitro Development of CD4+Lymphocytes with a Th2 Cytokine Profile

The Journal of Immunology ◽

10.4049/jimmunol.164.6.3047 ◽

2000 ◽

Vol 164 (6) ◽

pp. 3047-3055 ◽

Cited By ~ 193

Author(s):

Dragana Jankovic ◽

Marika C. Kullberg ◽

Nancy Noben-Trauth ◽

Patricia Caspar ◽

William E. Paul ◽

...

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Cytokine Profile ◽

Cell Analysis ◽

In Vitro Development ◽

Th2 Cytokine ◽

Vitro Development ◽

Cd4 Lymphocytes

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Effects of Ethanol and Acetaldehyde on Tight Junction Integrity: In Vitro Study in a Three Dimensional Intestinal Epithelial Cell Culture Model

PLoS ONE ◽

10.1371/journal.pone.0035008 ◽

2012 ◽

Vol 7 (4) ◽

pp. e35008 ◽

Cited By ~ 60

Author(s):

Elhaseen Elamin ◽

Daisy Jonkers ◽

Kati Juuti-Uusitalo ◽

Sven van IJzendoorn ◽

Freddy Troost ◽

...

Keyword(s):

Cell Culture ◽

Epithelial Cell ◽

Intestinal Epithelial Cell ◽

Three Dimensional ◽

In Vitro Study ◽

Intestinal Epithelial ◽

Cell Culture Model ◽

Epithelial Cell Culture ◽

Culture Model

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Single-cell analysis reveals IGF-1 potentiation of inhibition of the TGF-β/Smad pathway of fibrosis in human keratocytes in vitro

Scientific Reports ◽

10.1038/srep34373 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 11

Author(s):

Tomislav Sarenac ◽

Martin Trapecar ◽

Lidija Gradisnik ◽

Marjan Slak Rupnik ◽

Dusica Pahor

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Cell Analysis ◽

Smad Pathway ◽

Human Keratocytes

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Advances in Single-Cell Printing

Micromachines ◽

10.3390/mi13010080 ◽

2022 ◽

Vol 13 (1) ◽

pp. 80

Author(s):

Xiaohu Zhou ◽

Han Wu ◽

Haotian Wen ◽

Bo Zheng

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Three Dimensional ◽

Cell Isolation ◽

Special Focus ◽

Cell Analysis ◽

Cell Array ◽

Cell Printing ◽

Recent Developments ◽

Single Cell Isolation

Single-cell analysis is becoming an indispensable tool in modern biological and medical research. Single-cell isolation is the key step for single-cell analysis. Single-cell printing shows several distinct advantages among the single-cell isolation techniques, such as precise deposition, high encapsulation efficiency, and easy recovery. Therefore, recent developments in single-cell printing have attracted extensive attention. We review herein the recently developed bioprinting strategies with single-cell resolution, with a special focus on inkjet-like single-cell printing. First, we discuss the common cell printing strategies and introduce several typical and advanced printing strategies. Then, we introduce several typical applications based on single-cell printing, from single-cell array screening and mass spectrometry-based single-cell analysis to three-dimensional tissue formation. In the last part, we discuss the pros and cons of the single-cell strategies and provide a brief outlook for single-cell printing.

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Single-cell analysis revealed that IL4I1 promoted ovarian cancer progression

Journal of Translational Medicine ◽

10.1186/s12967-021-03123-7 ◽

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.

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HIV-1 provirus transcription and translation in macrophages differs from pre-integrated cDNA complexes and requires E2F transcriptional programs

10.1101/2021.04.21.440655 ◽

2021 ◽

Author(s):

Albebson L. Lim ◽

Philip Moos ◽

Christopher D. Pond ◽

Erica C. Larson ◽

Laura J. Martins ◽

...

Keyword(s):

Single Cell ◽

Single Cell Analysis ◽

Immunological Response ◽

Virus Production ◽

Cell Analysis ◽

Gene Transcripts ◽

Nuanced Understanding ◽

New Perspective ◽

Hiv 1

AbstractHIV-1 cDNA pre-integration complexes have been shown to persist for weeks in macrophages and to be transcriptionally active. Early and late gene transcripts are produced, along with some viral proteins, yet whole virus is not. While previous work has focused on the transcription and translation of HIV-1 genes; our understanding of cellular milieu that accompanies viral production is incomplete. We have used an in vitro system to model HIV-1 infection of macrophages, and single cell RNA sequencing (scRNA-seq) to compare the transcriptomes of uninfected cells, cells harboring pre-integration HIV-1 complexes (PIC) and those containing integrated provirus and actively making late HIV proteins. These are also compared to control cells, not exposed to virus.Several observations provide new perspective on the effects of HIV-1 transcription from pre-integrated cDNA versus from integrated provirus. First, HIV-1 transcript levels do not necessarily correlate with virus production, cells harboring PIC cDNA have transcript loads comparable to cells transcribing from provirus and making p24, mCherry, and vpu proteins. Second, all HIV-1 transcripts are easily detectable in abundance from PIC cDNA transcription, as is the case with cells transcribing from provirus, although the frequency of PIC cells with detectable gag-pol, tat, env, and nef transcripts is higher than the corresponding frequencies observed for “Provirus cells”. Third, the background transcriptomes of cells harboring pre- integrated HIV-1 cDNA are not otherwise detectably altered from cells not containing any HIV- 1 transcript. Fourth, integration and production of p24, mCherry, and Vpu proteins is accompanied by a switch from transcriptomes characterized by NFkB and AP-1 promoted transcription to a transcriptome characterized by E2F family transcription products. While some of these observations may seem heretical, single cell analysis provides a more nuanced understanding of PIC cDNA transcription and the transcriptomic changes that support HIV-1 protein production from integrated provirus.Author SummarySingle cell analysis is able to distinguish between HIV-1 infected macrophage cells that are transcribing pre-integrated HIV-1 cDNA and those transcribing HIV-1 provirus. Only cells transcribing HIV-1 provirus are making p24, marker mCherry and Vpu proteins, which corresponds with a change in the host cell’s background transcriptome from one expressing viral restriction and immunological response genes to one that is expressing genes associated with cell replication and oxidative phosphorylation.

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Studying human reproductive biology through single-cell analysis and in vitro differentiation of stem cells into germ cell-like cells

Human Reproduction Update ◽

10.1093/humupd/dmaa021 ◽

2020 ◽

Vol 26 (5) ◽

pp. 670-688 ◽

Cited By ~ 1

Author(s):

Lin Li ◽

Risako Yang ◽

Chenghong Yin ◽

Kehkooi Kee

Keyword(s):

Stem Cells ◽

Single Cell ◽

Germ Cells ◽

Regulatory Networks ◽

Single Cell Analysis ◽

Reproductive Development ◽

In Vitro Differentiation ◽

Gene Markers ◽

Human Reproductive

Abstract BACKGROUND Understanding the molecular and cellular mechanisms of human reproductive development has been limited by the scarcity of human samples and ethical constraints. Recently, in vitro differentiation of human pluripotent stem cells into germ cells and single-cell analyses have opened new avenues to directly study human germ cells and identify unique mechanisms in human reproductive development. OBJECTIVE AND RATIONALE The goal of this review is to collate novel findings and insightful discoveries with these new methodologies, aiming at introducing researchers and clinicians to the use of these tools to study human reproductive biology and develop treatments for infertility. SEARCH METHODS PubMed was used to search articles and reviews with the following main keywords: in vitro differentiation, human stem cells, single-cell analysis, spermatogenesis, oogenesis, germ cells and other key terms related to these subjects. The search period included all publications from 2000 until now. OUTCOMES Single-cell analyses of human gonads have identified many important gene markers at different developmental stages and in subpopulations of cells. To validate the functional roles of these gene markers, researchers have used the in vitro differentiation of human pluripotent cells into germ cells and confirmed that some genetic requirements are unique in human germ cells and are not conserved in mouse models. Moreover, transcriptional regulatory networks and the interaction of germ and somatic cells in gonads were elucidated in these studies. WIDER IMPLICATIONS Single-cell analyses allow researchers to identify gene markers and potential regulatory networks using limited clinical samples. On the other hand, in vitro differentiation methods provide clinical researchers with tools to examine these newly identify gene markers and study the causative effects of mutations previously associated with infertility. Combining these two methodologies, researchers can identify gene markers and networks which are essential and unique in human reproductive development, thereby producing more accurate diagnostic tools for assessing reproductive disorders and developing treatments for infertility.

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An In Vitro Lung System to Assess the Proinflammatory Hazard of Carbon Nanotube Aerosols

International Journal of Molecular Sciences ◽

10.3390/ijms21155335 ◽

2020 ◽

Vol 21 (15) ◽

pp. 5335

Author(s):

Hana Barosova ◽

Bedia Begum Karakocak ◽

Dedy Septiadi ◽

Alke Petri-Fink ◽

Vicki Stone ◽

...

Keyword(s):

Prolonged Exposure ◽

Three Dimensional ◽

Adverse Outcomes ◽

Alveolar Epithelial Cells ◽

Proinflammatory Response ◽

Multiwalled Carbon ◽

Alveolar Epithelial ◽

Culture Model ◽

Air Liquid Interface

In vitro three-dimensional (3D) lung cell models have been thoroughly investigated in recent years and provide a reliable tool to assess the hazard associated with nanomaterials (NMs) released into the air. In this study, a 3D lung co-culture model was optimized to assess the hazard potential of multiwalled carbon nanotubes (MWCNTs), which is known to provoke inflammation and fibrosis, critical adverse outcomes linked to acute and prolonged NM exposure. The lung co-cultures were exposed to MWCNTs at the air-liquid interface (ALI) using the VITROCELL® Cloud system while considering realistic occupational exposure doses. The co-culture model was composed of three human cell lines: alveolar epithelial cells (A549), fibroblasts (MRC-5), and macrophages (differentiated THP-1). The model was exposed to two types of MWCNTs (Mitsui-7 and Nanocyl) at different concentrations (2–10 μg/cm2) to assess the proinflammatory as well as the profibrotic responses after acute (24 h, one exposure) and prolonged (96 h, repeated exposures) exposure cycles. The results showed that acute or prolonged exposure to different concentrations of the tested MWCNTs did not induce cytotoxicity or apparent profibrotic response; however, suggested the onset of proinflammatory response.

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