Combinatorial labeling of single cells for gene expression cytometry

Science ◽  
2015 ◽  
Vol 347 (6222) ◽  
pp. 1258367 ◽  
Author(s):  
H. Christina Fan ◽  
Glenn K. Fu ◽  
Stephen P. A. Fodor
Keyword(s):  
Gene Expression ◽  
Single Cells ◽  
Digital Gene Expression ◽  
High Sensitivity ◽  
Single Experiment ◽  
Molecular Barcoding ◽  
Current Implementation ◽  
Digital Gene Expression Profile ◽  
Rare Cells

We present a technically simple approach for gene expression cytometry combining next-generation sequencing with stochastic barcoding of single cells. A combinatorial library of beads bearing cell- and molecular-barcoding capture probes is used to uniquely label transcripts and reconstruct the digital gene expression profile of thousands of individual cells in a single experiment without the need for robotics or automation. We applied the technology to dissect the human hematopoietic system and to characterize heterogeneous response to in vitro stimulation. High sensitivity is demonstrated by detection of low-abundance transcripts and rare cells. Under current implementation, the technique can analyze a few thousand cells simultaneously and can readily scale to 10,000s or 100,000s of cells.

scholarly journals Characterization of directed differentiation by high-throughput single-cell RNA-Seq

2014 ◽  
Author(s):  
Magali Soumillon ◽  
Davide Cacchiarelli ◽  
Stefan Semrau ◽  
Alexander van Oudenaarden ◽  
Tarjei S Mikkelsen
Keyword(s):  
Gene Expression ◽  
Disease Modeling ◽  
Single Cells ◽  
Adipogenic Differentiation ◽  
Digital Gene Expression ◽  
Multiple Time ◽  
Directed Differentiation ◽  
Time Points

Directed differentiation of cells in vitro is a powerful approach for dissection of developmental pathways, disease modeling and regenerative medicine, but analysis of such systems is complicated by heterogeneous and asynchronous cellular responses to differentiation-inducing stimuli. To enable deep characterization of heterogeneous cell populations, we developed an efficient digital gene expression profiling protocol that enables surveying of mRNA in thousands of single cells at a time. We then applied this protocol to profile 12,832 cells collected at multiple time points during directed adipogenic differentiation of human adipose-derived stem/stromal cells in vitro. The resulting data reveal the major axes of cell-to-cell variation within and between time points, and an inverse relationship between inflammatory gene expression and lipid accumulation across cells from a single donor.

scholarly journals ZipSeq : Barcoding for Real-time Mapping of Single Cell Transcriptomes

2020 ◽  
Author(s):  
Kenneth H. Hu ◽  
John P. Eichorst ◽  
Chris S. McGinnis ◽  
David M. Patterson ◽  
Eric D. Chow ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
Real Time ◽  
Dimensional Space ◽  
Single Cells ◽  
Expression Patterns ◽  
Live Cells ◽  
Glass Substrates ◽  
Complete Mapping

ABSTRACTSpatial transcriptomics seeks to integrate single-cell transcriptomic data within the 3-dimensional space of multicellular biology. Current methods use glass substrates pre-seeded with matrices of barcodes or fluorescence hybridization of a limited number of probes. We developed an alternative approach, called ‘ZipSeq’, that uses patterned illumination and photocaged oligonucleotides to serially print barcodes (Zipcodes) onto live cells within intact tissues, in real-time and with on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in-vitro wound healing, live lymph node sections and in a live tumor microenvironment (TME). In all cases, we discovered new gene expression patterns associated with histological structures. In the TME, this demonstrated a trajectory of myeloid and T cell differentiation, from periphery inward. A variation of ZipSeq efficiently scales to the level of single cells, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.

scholarly journals Expression of Genes Related to Germ Cell Lineage and Pluripotency in Single Cells and Colonies of Human Adult Germ Stem Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-17 ◽  
Author(s):  
Sabine Conrad ◽  
Hossein Azizi ◽  
Maryam Hatami ◽  
Mikael Kubista ◽  
Michael Bonin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Gene Expression Profile ◽  
Expression Profile ◽  
Adult Stem Cells ◽  
Single Cells ◽  
Human Fibroblasts ◽  
Embryonic Stem ◽  
Human Adult

The aim of this study was to elucidate the molecular status of single human adult germ stem cells (haGSCs) and haGSC colonies, which spontaneously developed from the CD49f MACS and matrix- (collagen−/laminin+ binding-) selected fraction of enriched spermatogonia. Single-cell transcriptional profiling by Fluidigm BioMark system of a long-term cultured haGSCs cluster in comparison to human embryonic stem cells (hESCs) and human fibroblasts (hFibs) revealed that haGSCs showed a characteristic germ- and pluripotency-associated gene expression profile with some similarities to hESCs and with a significant distinction from somatic hFibs. Genome-wide comparisons with microarray analysis confirmed that different haGSC colonies exhibited gene expression heterogeneity with more or less pluripotency. The results of this study confirm that haGSCs are adult stem cells with a specific molecular gene expression profilein vitro, related but not identical to true pluripotent stem cells. Under ES-cell conditions haGSC colonies could be selected and maintained in a partial pluripotent state at the molecular level, which may be related to their cell plasticity and potential to differentiate into cells of all germ layers.

scholarly journals Memory sequencing reveals heritable single cell gene expression programs associated with distinct cellular behaviors

2018 ◽  
Author(s):  
Sydney M. Shaffer ◽  
Benjamin L. Emert ◽  
Raul Reyes-Hueros ◽  
Christopher Coté ◽  
Guillaume Harmange ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cells ◽  
Cancer Therapeutics ◽  
Population Based ◽  
Fluctuation Analysis ◽  
Genetic Heritability ◽  
The Face ◽  
Cell Gene Expression ◽  
Cell Subpopulations ◽  
Rare Cells

AbstractNon-genetic factors can cause individual cells to fluctuate substantially in gene expression levels over time. Yet it remains unclear whether these fluctuations can persist for much longer than the time of one cell division. Current methods for measuring gene expression in single cells mostly rely on single time point measurements, making the duration of gene expression fluctuations or cellular memory difficult to measure. Here, we report a method combining Luria and Delbrück’s fluctuation analysis with population-based RNA sequencing (MemorySeq) for identifying genes transcriptome-wide whose fluctuations persist for several cell divisions. MemorySeq revealed multiple gene modules that are expressed together in rare cells within otherwise homogeneous clonal populations. Further, we found that these rare cell subpopulations are associated with biologically distinct behaviors, such as the ability to proliferate in the face of anti-cancer therapeutics, in different cancer cell lines. The identification of non-genetic, multigenerational fluctuations has the potential to reveal new forms of biological memory at the level of single cells and suggests that non-genetic heritability of cellular state may be a quantitative property.

scholarly journals High Proliferative Placenta-Derived Multipotent Cells Express Cytokeratin 7 at Low Level

2019 ◽  
Vol 2019 ◽  
pp. 1-13
Author(s):  
V. Shablii ◽  
M. Kuchma ◽  
H. Svitina ◽  
I. Skrypkina ◽  
P. Areshkov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cells ◽  
Placental Tissue ◽  
First Trimester ◽  
Mesenchymal Cells ◽  
Culture Conditions ◽  
Multipotent Cells ◽  
Genes Expression

The purpose of this study was to investigate the immunophenotypes and gene expression profile of high proliferative placenta-derived multipotent cells (PDMCs) population at different stages of culture. We demonstrated that the colonies resulting from single cells were either positive or negative for CK7, whereas only PDMC clones with weak CK7 expression (CK7low-clones) were highly proliferative. Interestingly, vimentin positive (Vim+) placental stromal mesenchymal cells did not express CK7 in situ, but double CK7+Vim+ cells detection in tissue explants and explants outgrowth indicated CK7 inducible expression in vitro. PCNA presence in CK7+Vim+ cells during placental explants culturing confirmed belonging of these cells to proliferative subpopulation. Transcription factors CDX2 and EOMES were expressed in both CK7low-clones and subset of stromal mesenchymal cells of first-trimester placental tissue in situ. Meanwhile, CK7low -clones and stromal mesenchymal cells of full-term placental tissue in situ expressed ERG heterogeneously. SPP1, COL2A1, and PPARG2 mesodermal-related genes expression by CK7low-clones additionally confirms their mesenchymal origin. Inherent stem cell-related gene expression (IFTM3, POU5F1, and VASA) in CK7low-clones might indicate their enrichment for progenitors. Finally, in CK7low-clones we observed expression of such trophoblast-associated genes as CGB types I and II, fusogenic ERVW-1, GCM1, and GATA3. Thus, our results indicate that PDMCs acquired the representative immunophenotype signature under culture conditions.

scholarly journals Screening and analysis on the differentially expression genes between diploid and autotetraploid watermelon by using of digital gene expression profile

2019 ◽  
Vol 79 (2) ◽  
pp. 180-190 ◽  
Author(s):  
Y. L. Long ◽  
F. Qiao ◽  
X. F. Jiang ◽  
H. Q. Cong ◽  
M. L. Sun ◽  
...  
Keyword(s):  
Gene Expression ◽  
Expression Patterns ◽  
Digital Gene Expression ◽  
Background Level ◽  
Global Gene Expression ◽  
Gene Expression Patterns ◽  
Protein Signaling ◽  
Genome Doubling ◽  
Digital Gene Expression Profile ◽  
Stress Related Genes

Abstract Synthetic polyploids are key breeding materials for watermelon. Compared with diploid watermelon, the tetraploid watermelon often exhibit wide phenotypic differences and differential gene expression. Digital gene expression (DGE) profile technique was performed in this study to present gene expression patterns in an autotetraploid and its progenitor diploid watermelon, and deferentially expressed genes (DEGs) related to the abiotic and biotic stress were also addressed. Altogether, 4,985 DEGs were obtained in the autotetraploid against its progenitor diploid, and 66.02% DEGs is up-regulated. GO analysis shows that these DEGs mainly distributed in ‘metabolic process’, ‘cell’ and ‘catalytic activity’. KEGG analysis revealed that these DEGs mainly cover ‘metabolic pathways’, ‘secondary metabolites’ and ‘ribosome’. Moreover, 134 tolerance related DEGs were identified which cover osmotic adjustment substance, protective enzymes/protein, signaling proteins and pathogenesis-related proteins. This study present the differential expression of stress related genes and global gene expression patterns at background level in autotetraploid watermelons. These new evidences could supplement the molecular theoretical basis for the better resistance after the genome doubling in the gourd family.

279 DIFFERENCES IN TRANSCRIPT ABUNDANCE BETWEEN MALE AND FEMALE BOVINE BLASTOCYSTS PRODUCED IN VITRO USING SEXED SEMEN

2007 ◽  
Vol 19 (1) ◽  
pp. 255
Author(s):  
D. Rizos ◽  
P. Lonergan ◽  
D. Rath ◽  
J. De la Fuente ◽  
M. Wade ◽  
...  
Keyword(s):  
Gene Expression ◽  
Relative Abundance ◽  
Dna Methyltransferase ◽  
High Sensitivity ◽  
Transcript Abundance ◽  
Culture Conditions ◽  
Interferon Tau ◽  
Fluid Medium ◽  
Male And Female

There is considerable evidence in the literature indicating that, depending on the culture environment, male pre-implantation embryos develop faster than females, with, for example, a higher proportion of early developing blastocysts being male. The aim of this study was to examine gender-related differences in gene expression in bovine blastocysts produced in vitro following IVF with sex-sorted, frozen–thawed semen. For blastocyst production, immature cumulus–oocyte complexes (COCs) recovered from the ovaries of slaughtered heifers were matured in vitro for 24 h. Matured COCs were randomly split into 2 groups and inseminated with frozen–thawed sperm sorted flow cytometrically for gender. Presumptive zygotes were cultured for 7 days in synthetic oviduct fluid medium. In a preliminary experiment, blastocysts (XX: n = 61; XY: n = 47) were recovered on Day 8 post-insemination and individually snap frozen in liquid nitrogen for verification of the sorting procedure. Sexing was performed with PCR using both male-specific (BRY4.a) and bovine satellite primers. The proportion of female and male blastocysts obtained with X- and Y-chromosome-bearing sperm was 87.2 and 80.3%, respectively. In a subsequent 5 replicates, Day 8 blastocysts were snap frozen in groups of 10 for analysis of mRNA relative abundance. These developmentally important genes were selected based on microarray analysis because they showed a high sensitivity to suboptimal in vitro culture conditions (data not shown). There was no difference in the relative abundance of desmocollin II (DcII), Na/K-ATPase alpha1 subunit (Na/K), ubiquitin-activating enzyme E2 (Ube2), fibroblast growth factor 4 (FGF4), and DNA methyltransferase 1 (Dnmt1) between male and female blastocysts. X-inactive specific transcript (Xist), interferon-tau (IFN), and a Sry-related HMG box transcriptoinal factor (Sox17) were significantly up-regulated in female blastocysts, whereas DNA methyltransferase 3a (Dnmt3a), DNA methyltransferase 3b (Dnmt3b), sarcosine oxidase (Sox), and the transcription factor Oct-4 were significantly up-regulated in males. In conclusion, differences in gene expression between male and female embryos exist and may be related to the well-described differences in the kinetics of development.

Hoxa9+Meis1a Efficiently Transform Hematopoietic Stem Cells but Not Committed Progenitors.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3375-3375
Author(s):  
Yingzi Wang ◽  
Andrei Krivtsov ◽  
Grigoriy Losyev ◽  
Scott A. Armstrong
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
High Efficiency ◽  
Single Cells ◽  
Global Gene Expression ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Clonogenic Potential

Abstract The homeobox transcription factors Hoxa9 and Meis1a induce the expansion of hematopoietic stem cells (HSC) and cooperate to induce acute myeloid leukemia (AML). We have recently shown that Hoxa9 and Meis1a are part of a self-renewal program found in leukemia stem cells (LSC) isolated from murine leukemias initiated by expression of MLL-AF9 in committed granulocyte macrophage progenitors (GMP). However, it remains unclear whether the Hoxa9-Meis1a complex is sufficient to mimic the leukemogenic effects of MLL-AF9. Therefore we assessed the clonogenic and leukemogenic activity of HSC and GMP transduced with Hoxa9-Meis1a and compared this to MLL-AF9 transduced HSC and GMP. We expressed either Hoxa9-Meis1a or MLL-AF9 in HSC or GMP and sorted single cells into 96 well plates. The data demonstrate that the clonogenic potential of single HSC or GMP expressing Hoxa9-Meis1a or MLL-AF9 are similar and both produce cells that can be replated in vitro for greater than 6 weeks. Remarkably, Hoxa9-Meis1a induces leukemia with high efficiency when expressed in HSC but not committed GMP, while MLL-AF9 can fully transform both HSC and GMP. Next, we identified a population of cells from Hoxa9-Meis1a leukemias that were enriched for LSC. Even though the leukemias were initiated from HSC, the LSC population possessed an immunophenotype and global gene expression program more consistent with differentiated myeloid cells. Further characterization of gene expression in the Hoxa9-Meis1a LSC found that only a subset of the MLL-AF9 self-renewal signature is activated by Hoxa9-Meis1a. These findings show that Hoxa9-Meis1a can efficiently induce LSC from HSC but not GMP. This suggests Hoxa9 and Meis1a induced leukemogenesis is dependent upon cellular context and require programs/pathways active in HSC in order to initiate leukemia. Thus MLL-AF9 must activate pathways critical for LSC development in addition to Hoxa9-Meis1a in order to fully transform committed progenitor cells. Identification of these cooperating pathways should provide insight into MLL-rearranged and other AML.

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