Mouse intestinal organoid time-course experiments from single cells

Abstract Organoids recapitulate the self-organizing capacity of stem cells and the tissue organization of the original organ in a controlled and trackable environment. Intestinal organoids, in particular, can develop from a single cell into a fully-grown structure that contains most of the cell types, patterns, and morphogenetic properties of the adult intestine. Here we present a protocol for high-throughput organoid culture in multi-well plate format combined with high content immunofluorescence imaging and RNA extraction. Our protocol allows recording and analysis of thousands of organoids during several days of development.

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Microfluidic guillotine for single-cell wound repair studies

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705059114 ◽

2017 ◽

Vol 114 (28) ◽

pp. 7283-7288 ◽

Cited By ~ 14

Author(s):

Lucas R. Blauch ◽

Ya Gai ◽

Jian Wei Khor ◽

Pranidhi Sood ◽

Wallace F. Marshall ◽

...

Keyword(s):

Single Cell ◽

High Throughput ◽

Wound Repair ◽

Time Course ◽

Single Cells ◽

Repair Process ◽

Gene Knockdown ◽

Viscous Stress ◽

Repair Capacity ◽

Stentor Coeruleus

Wound repair is a key feature distinguishing living from nonliving matter. Single cells are increasingly recognized to be capable of healing wounds. The lack of reproducible, high-throughput wounding methods has hindered single-cell wound repair studies. This work describes a microfluidic guillotine for bisecting single Stentor coeruleus cells in a continuous-flow manner. Stentor is used as a model due to its robust repair capacity and the ability to perform gene knockdown in a high-throughput manner. Local cutting dynamics reveals two regimes under which cells are bisected, one at low viscous stress where cells are cut with small membrane ruptures and high viability and one at high viscous stress where cells are cut with extended membrane ruptures and decreased viability. A cutting throughput up to 64 cells per minute—more than 200 times faster than current methods—is achieved. The method allows the generation of more than 100 cells in a synchronized stage of their repair process. This capacity, combined with high-throughput gene knockdown in Stentor, enables time-course mechanistic studies impossible with current wounding methods.

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Construction of 3D Cellular Composites with Stem Cells Derived from Adipose Tissue and Endothelial Cells by Use of Optical Tweezers in a Natural Polymer Solution

Materials ◽

10.3390/ma12111759 ◽

2019 ◽

Vol 12 (11) ◽

pp. 1759 ◽

Cited By ~ 1

Author(s):

Takehiro Yamazaki ◽

Toshifumi Kishimoto ◽

Paweł Leszczyński ◽

Koichiro Sadakane ◽

Takahiro Kenmotsu ◽

...

Keyword(s):

Stem Cells ◽

Endothelial Cells ◽

Adipose Tissue ◽

Optical Tweezers ◽

Single Cells ◽

Three Dimensional ◽

Cell Types ◽

Cellular Interactions ◽

Research Fields ◽

Wide Range

To better understand the regulation and function of cellular interactions, three-dimensional (3D) assemblies of single cells and subsequent functional analysis are gaining popularity in many research fields. While we have developed strategies to build stable cellular structures using optical tweezers in a minimally invasive state, methods for manipulating a wide range of cell types have yet to be established. To mimic organ-like structures, the construction of 3D cellular assemblies with variety of cell types is essential. Our recent studies have shown that the presence of nonspecific soluble polymers in aqueous solution is the key to creating stable 3D cellular assemblies efficiently. The present study further expands on the construction of 3D single cell assemblies using two different cell types. We have successfully generated 3D cellular assemblies, using GFP-labeled adipose tissue-derived stem cells and endothelial cells by using optical tweezers. Our findings will support the development of future applications to further characterize cellular interactions in tissue regeneration.

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Simultaneous tracking of division and differentiation from individual hematopoietic stem and progenitor cells reveals within-family homogeneity despite population heterogeneity

10.1101/586354 ◽

2019 ◽

Cited By ~ 1

Author(s):

Tamar Tak ◽

Giulio Prevedello ◽

Gaël Simon ◽

Noémie Paillon ◽

Ken R. Duffy ◽

...

Keyword(s):

Progenitor Cells ◽

High Throughput ◽

Common Ancestor ◽

Single Cells ◽

Population Level ◽

Cell Types ◽

Culture Conditions ◽

Population Heterogeneity ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

AbstractThe advent of high throughput single cell methods such as scRNA-seq has uncovered substantial heterogeneity in the pool of hematopoietic stem and progenitor cells (HSPCs). A significant issue is how to reconcile those findings with the standard model of hematopoietic development, and a fundamental question is how much instruction is inherited by offspring from their ancestors. To address this, we further developed a high-throughput method that enables simultaneously determination of common ancestor, generation, and differentiation status of a large collection of single cells. Data from it revealed that while there is substantial population-level heterogeneity, cells that derived from a common ancestor were highly concordant in their division progression and share similar differentiation outcomes, revealing significant familial effects on both division and differentiation. Although each family diversifies to some extent, the overall collection of cell types observed in a population is largely composed of homogeneous families from heterogeneous ancestors. Heterogeneity between families could be explained, in part, by differences in ancestral expression of cell-surface markers that are used for phenotypic HSPC identification: CD48, SCA-1, c-kit and Flt3. These data call for a revision of the fundamental model of haematopoiesis from a single tree to an ensemble of trees from distinct ancestors where common ancestor effect must be considered. As HSPCs are cultured in the clinic before bone marrow transplantation, our results suggest that the broad range of engraftment and proliferation capacities of HSPCs could be consequences of the heterogeneity in their engrafted families, and altered culture conditions might reduce heterogeneity between families, possibly improving transplantation outcomes.

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Single-Cell RNA Sequencing to Disentangle the Blood System

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvbaha.120.314654 ◽

2021 ◽

Vol 41 (3) ◽

pp. 1012-1018

Author(s):

Jean Acosta ◽

Daniel Ssozi ◽

Peter van Galen

Keyword(s):

Blood Cell ◽

Single Cell ◽

Rna Sequencing ◽

Molecular Mechanisms ◽

Single Cells ◽

Cell Types ◽

Blood System ◽

Differentiated Cells ◽

Tissue Organization ◽

Single Cell Rna Sequencing

The blood system is often represented as a tree-like structure with stem cells that give rise to mature blood cell types through a series of demarcated steps. Although this representation has served as a model of hierarchical tissue organization for decades, single-cell technologies are shedding new light on the abundance of cell type intermediates and the molecular mechanisms that ensure balanced replenishment of differentiated cells. In this Brief Review, we exemplify new insights into blood cell differentiation generated by single-cell RNA sequencing, summarize considerations for the application of this technology, and highlight innovations that are leading the way to understand hematopoiesis at the resolution of single cells. Graphic Abstract: A graphic abstract is available for this article.

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7UTILIZATION OF CELL PROFILING TO EVALUATE BOVINE SPERMATOZOA IN NORMAL AND SIMULATED MICROGRAVITY

Reproduction Fertility and Development ◽

10.1071/rdv16n1ab7 ◽

2004 ◽

Vol 16 (2) ◽

pp. 126

Author(s):

J.C. Andrews ◽

S. Winters-Hilt

Keyword(s):

Time Course ◽

Simulated Microgravity ◽

Single Cells ◽

Mrna Translation ◽

Cell Types ◽

Support Vector ◽

Mitochondrial Activity ◽

C Programs ◽

Mammalian Spermatozoon ◽

Bovine Spermatozoa

We developed a method to evaluate bovine sperm membranes in normal (1G) and simulated microgravity (Sim-μG). Bovine spermatozoa are used as a model system because they have cellular membranes analogous to those of other cell types, and yet are much simpler because they have no cytoplasm and do not participate in DNA transcription or mRNA translation. They can be cultured as single cells and are easily evaluated for membrane characteristics using flow cytometry. These features make the mammalian spermatozoon a useful model for exploring the principles of membrane structure/function in the presence of a variety of environmental challenges such as simulated microgravity. Cryopreserved, washed beef bull sperm (4–8×106mL−1)were incubated under non-capacitating conditions (modified glucose-free Tyrode’s medium containing low bicarbonate, HEPES buffer, pyruvate and 3mgmL−1 BSA V; 23°C in air), and these spermatozoa remained alive for 24–48h at 1G. To simulate μG, spermatozoa were incubated under the same conditions, in a HARV 10 rotating wall vessel (RWV, Synthecon, Inc, Houston, TX, USA) at 9rpms. Spermatozoa were incubated in 1G and Sim-μG environments for 2.5–4.5h and subsequently exposed to 0, 60 or 80μgmL−1 LC for 0, 4, 8, 12, 16 and 20min. Three fluorochrome combinations were used as probes at each [LC]/time point: (1) propidium Iodide (dead status)+SYBR 14 (live status); (2) PI+FITC-PSA (acrosome reactions [ARs]); (3) PI+MitoTracker Deep Red (mitochondrial activity). Approximately 1million spermatozoa from 3 bulls were evaluated over 4 days. Data were acquired on a FACSVantage SE flow cytometer, and initially analyzed (quality control) using the bundled FACSVantage SE software package (Cell Quest, BD BioSciences, San Jose, CA, USA). This provided graphics of simple cell relations (fluorescence v. LC exposure time). For further statistical analysis, and incorporation of non-parametric statistical tools (including pattern recognition using Support Vector Machines), the data were processed using a collection of Perl scripts and C programs. Results: Live/dead status: When Sim-μG+60μgmL−1 LC sperm were compared to 1G+60μgmL−1LC, and 80μgmL−1 LC sperm, their profiles were more similar to the 1G 80μgmL−1 LC profiles. AR status: the Sim-μG+60μgmL−1 LC profiles were similar to the 1G+60μgmL−1 LC profiles. Mitochondrial Status: the Sim-μG+60μgmL−1LC profiles were more similar to 1G+80μgmL−1 LC profiles. Summary: although Sim-μG sperm lost their motility within 3h, they were alive. Cell profiles indicate that Sim-μG sperm nuclear membranes are less stable and their mitochondria are less functional than the 1G controls, but their acrosomes are intact indicating that fertilizing potential may remain. Additional experiments are needed to determine the time course for Sim-μG, induced changes, and whether Sim-μG sperm can penetrate eggs. Funding: NASA (2002)-Stennis-24 and The University of New Orleans.

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RNA splicing programs define tissue compartments and cell types at single cell resolution

10.1101/2021.05.01.442281 ◽

2021 ◽

Author(s):

Julia Eve Olivieri ◽

Roozbeh Dehghannasiri ◽

Peter Wang ◽

SoRi Jang ◽

Antoine de Morree ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

High Throughput ◽

Rna Splicing ◽

Single Cells ◽

Cell Types ◽

Mouse Lemur ◽

Cell Type ◽

Multiple Organs ◽

Single Cell Pcr

More than 95% of human genes are alternatively spliced. Yet, the extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach that is agnostic to transcript annotation, to detect cell-type-specific regulated splicing in > 110K carefully annotated single cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type specifically spliced. These results are validated with RNA FISH, single cell PCR, and in high throughput with Smart-seq2. Regulated splicing is found in ubiquitously expressed genes such as actin light chain subunit MYL6 and ribosomal protein RPS24, which has an epithelial-specific microexon. 13% of the statistically most variable splice sites in cell-type specifically regulated genes are also most variable in mouse lemur or mouse. SpliZ analysis further reveals 170 genes with regulated splicing during sperm development using, 10 of which are conserved in mouse and mouse lemur. The statistical properties of the SpliZ allow model-based identification of subpopulations within otherwise indistinguishable cells based on gene expression, illustrated by subpopulations of classical monocytes with stereotyped splicing, including an un-annotated exon, in SAT1, a Diamine acetyltransferase. Together, this unsupervised and annotation-free analysis of differential splicing in ultra high throughput droplet-based sequencing of human cells across multiple organs establishes splicing is regulated cell-type-specifically independent of gene expression.

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The CD11a and EPCR marker combination simplifies and improves the purification of mouse hematopoietic stem cells

10.1101/219063 ◽

2017 ◽

Author(s):

Alborz Karimzadeh ◽

Vanessa Scarfone ◽

Connie Chao ◽

Karin Grathwohl ◽

John W. Fathman ◽

...

Keyword(s):

Stem Cells ◽

Hematopoietic Stem Cells ◽

Gold Standard ◽

Cell Types ◽

The Self ◽

Mouse Bone Marrow ◽

Surface Markers ◽

Hematopoietic Stem ◽

Multipotent Progenitors ◽

Marker Combination

AbstractHematopoietic stem cells (HSCs) are the self-renewing multipotent progenitors to all blood cell types. Identification and isolation of HSCs for study has depended on the expression of combinations of surface markers on HSCs that reliably distinguish it from other cell types. However, the increasing number of markers required to isolate HSCs has made it tedious, expensive, and difficult for newcomers, suggesting the need for a simpler panel of HSC markers. We previously showed that phenotypic HSCs could be separated based on expression of CD11a, and that only the CD11a negative fraction contained true HSCs. Here, we show that CD11a and another HSC marker, EPCR, can be used to effectively identify and purify HSCs. We introduce a new two-color HSC sorting method that can highly enrich for HSCs with efficiencies comparable to the gold standard combination of CD150 and CD48. Our results demonstrate that adding CD11a and EPCR to the HSC biologist’s toolkit improves the purity of and simplifies isolation of HSCs.Significance StatementThe study of hematopoietic stem cells (HSCs) and their purification for transplantation requires a panel of surface markers that can be used to distinguish HSCs from other cell types. The number of markers necessary to identify HSCs continues to grow, making it increasingly difficult to identify HSCs by flow cytometry. In this study, we identified a combination of two surface markers, CD11a and EPCR, to enrich for HSCs in the mouse bone marrow without the need for additional markers. This simplified panel could aid HSC research by reducing the number of markers necessary to identify and isolate HSCs.

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Single-cell phenotyping of human induced pluripotent stem cells by high-throughput imaging

10.1101/026955 ◽

2015 ◽

Cited By ~ 2

Author(s):

Hans Christian Volz ◽

Florian Heigwer ◽

Tatjana Wuest ◽

Marta Galach ◽

Jochen Utikal ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Single Cell ◽

High Throughput ◽

Pluripotent Stem Cells ◽

Single Cells ◽

Phenotypic Variability ◽

Screening Experiments ◽

Induced Pluripotent ◽

High Throughput Imaging

Single-cell phenotyping promises to yield insights into biological responses in heterogeneous cell populations. We developed a method based on single-cell analysis to phenotype human induced pluripotent stem cells (hIPSC) by high-throughput imaging. Our method uses markers for morphology and pluripotency as well as social features to characterize perturbations using a meta-phenotype based on mapping single cells to distinct phenotypic classes. Analysis of perturbations on a single cell level enhances the applicability of human induced pluripotent stem cells (hIPSC) for screening experiments taking the inherently increased phenotypic variability of these cells into account. We adapted miniaturized culture conditions to allow for the utilization of hIPSC in RNA interference (RNAi) high-throughput screens and single cell phenotyping by image analysis. We identified key regulators of pluripotency in hIPSC masked in a population-averaged analysis and we confirmed several candidate genes (SMG1, TAF1) and assessed their effect on pluripotency.

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STEM-27. A PERIVASCULAR STEM CELL UNDERLIES VERTEBRATE MENINGEAL TUMORIGENESIS

Neuro-Oncology ◽

10.1093/neuonc/noab196.101 ◽

2021 ◽

Vol 23 (Supplement_6) ◽

pp. vi26-vi27

Author(s):

Abrar Choudhury ◽

Martha Cady ◽

Calixto Lucas ◽

Brisa Palikuqi ◽

Ophir Klein ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Single Cell ◽

Tumor Cells ◽

Rna Sequencing ◽

Single Cells ◽

Cell Types ◽

Human Meningioma ◽

Single Cell Rna Sequencing ◽

Human Meningiomas

Abstract BACKGROUND Meningiomas are the most common primary intracranial tumors in humans and dogs, but biologic drivers and cell types underlying meningeal tumorigenesis are incompletely understood. Here we integrate meningioma single-cell RNA sequencing with stem cell approaches to define a perivascular stem cell underlying vertebrate meningeal tumorigenesis. METHODS Single-cell RNA sequencing was performed on 57,114 cells from 8 human meningiomas, 54,607 cells from 3 dog meningiomas, and human meningioma xenografts in mice. Results were validated using immunofluorescence (IF), immunohistochemistry (IHC), and deconvolution of bulk RNA sequencing of 200 human meningiomas. Mechanistic and functional studies were performed using clonogenic and limiting dilution assays, xenografts, and genetically engineered mouse models. RESULTS Copy number variant identification from human meningioma single cells distinguished tumor cells with loss of chr22q from non-tumor cells with intact chr22q. A single cluster distinguished by expression of Notch3 and other cancer stem cell genes had an intermediate level of loss of chr22q, suggesting this cluster may represent meningioma stem cells. In support of this hypothesis, pseudotime trajectory analysis demonstrated transcriptomic progression starting from Notch3+ cells and encompassing all other meningioma cell types. Notch3+ meningioma cells had transcriptomic concordance to mural pericytes, and IF/IHC of prenatal and adult human meninges, as well as lineage tracing using a Notch3-CreERT2 allele in mice, confirmed Notch3+ cells were restricted to the perivascular stem cell niche in mammalian meningeal development and homeostasis. Integrating human and dog meningioma single cells revealed Notch3+ cells in tumor and non-tumor clusters in dog meningiomas. Notch3 IF/IHC and cell-type deconvolution of bulk RNA sequencing showed Notch3+ cells were enriched in high-grade human meningiomas. Notch3 overexpression in human meningioma cells increased clonogenic growth in vitro, and increased tumorigenesis and tumor growth in vivo, decreasing overall survival. CONCLUSIONS Notch3+ stem cells in the perivascular niche underlie vertebrate meningeal tumorigenesis.

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Cell contact induces multiple types of electrical excitability from ascidian two-cell embryos that are cleavage arrested and contain all cell fate determinants

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00835.2006 ◽

2007 ◽

Vol 293 (5) ◽

pp. R1976-R1996

Author(s):

Motoko Tanaka-Kunishima ◽

Kunitaro Takahashi ◽

Fumiyuki Watanabe

Keyword(s):

Stem Cells ◽

Cell Fate ◽

Single Cells ◽

Muscle Cells ◽

Cell Types ◽

Specific Cell ◽

Cell Type ◽

Cell Pair ◽

Fate Determinants ◽

Cell Fate Determinants

Ascidian early embryonic cells undergo cell differentiation without cell cleavage, thus enabling mixture of cell fate determinants in single cells, which will not be possible in mammalian systems. Either cell in a two-cell embryo (2C cell) has multiple fates and develops into any cell types in a tadpole. To find the condition for controlled induction of a specific cell type, cleavage-arrested cell triplets were prepared in various combinations. They were 2C cells in contact with a pair of anterior neuroectoderm cells from eight-cell embryos (2C-aa triplet), with a pair of presumptive notochordal neural cells (2C-AA triplet), with a pair of presumptive posterior epidermal cells (2C-bb triplet), and with a pair of presumptive muscle cells (2C-BB triplet). The fate of the 2C cell was electrophysiologically identified. When two-cell embryos had been fertilized 3 h later than eight-cell embryos and triplets were formed, the 2C cells became either anterior-neuronal, posterior-neuronal or muscle cells, depending on the cell type of the contacting cell pair. When two-cell embryos had been fertilized earlier than eight-cell embryos, most 2C cells became epidermal. When two- and eight-cell embryos had been simultaneously fertilized, the 2C cells became any one of three cell types described above or the epidermal cell type. Differentiation of the ascidian 2C cell into major cell types was reproducibly induced by selecting the type of contacting cell pair and the developmental time difference between the contacting cell pair and 2C cell. We discuss similarities between cleavage-arrested 2C cells and vertebrate embryonic stem cells and propose the ascidian 2C cell as a simple model for toti-potent stem cells.

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