scholarly journals Crucial stem cell experiments? Stem cells, uncertainty, and single-cell experiments

2015 ◽  
Vol 30 (2) ◽  
pp. 183
Author(s):  
Melinda Fagan
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Single Cells ◽  
Single Cell Level ◽  
Epistemic Circularity ◽  
Cell Level ◽  
Differentiation Potential ◽  
Self Renewal

I have previously argued that stem cell experiments cannot in principle demonstrate that a single cell is a stem cell ([reference omitted for anonymous review]).  Laplane and others dispute this claim, citing experiments that identify stem cells at the single-cell level.  This paper rebuts the counterexample, arguing that these alleged ‘crucial stem cell experiments’ do not measure self-renewal for a single cell, do not establish a single cell’s differentiation potential, and, if interpreted as providing results about single cells, fall into epistemic circularity.  I then examine the source of the dispute, noting differences in philosophical and experimental perspectives.

scholarly journals Single-cell transcriptome provides novel insights into antler stem cells, a cell type capable of mammalian organ regeneration

2018 ◽  
Author(s):  
Hengxing Ba ◽  
Datao Wang ◽  
Weiyao Wu ◽  
Hongmei Sun ◽  
Chunyi Li
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Large Cell ◽  
Stem Cell Markers ◽  
Single Cell Level ◽  
Cell Type ◽  
Cell Level ◽  
Homogeneous Population ◽  
Organ Regeneration

AbstractAntler regeneration, a stem cell-based epimorphic process, has potential as a valuable model for regenerative medicine. A pool of antler stem cells (ASCs) for antler development is located in the antlerogenic periosteum (AP). However, whether this ASC pool is homogenous or heterogeneous has not been fully evaluated. In this study, we produced a comprehensive transcriptome dataset at the single-cell level for the ASCs based on the 10x Genomics platform (scRNA-seq). A total of 4,565 ASCs were sequenced and classified into a large cell cluster, indicating that the ASCs resident in the AP are likely to be a homogeneous population. The scRNA-seq data revealed that tumor-related genes were highly expressed in these homogeneous ASCs: i.e. TIMP1, TMSB10, LGALS1, FTH1, VIM, LOC110126017 and S100A4. Results of screening for stem cell markers suggest that the ASCs may be considered as a special type of stem cell between embryonic (CD9) and adult (CD29, CD90, NPM1 and VIM) stem cells. Our results provide the first comprehensive transcriptome analysis at the single-cell level for the ASCs, and identified only one major cell type resident in the AP and some key stem cell genes, which may hold the key to why antlers, the unique mammalian organ, can fully regenerate once lost.

scholarly journals NRASG12V-Mediated Self-Renewal Signaling in Self-Renewing AML Stem Cells.

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2626-2626
Author(s):  
Marie Lue Antony ◽  
Klara Noble-Orcutt ◽  
Karen Sachs ◽  
Anna Khoroshilov ◽  
Daniel Chang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Molecular Mechanisms ◽  
Signaling Molecules ◽  
The Self ◽  
Single Cell Level ◽  
Mass Cytometry ◽  
Cell Level ◽  
Self Renewal ◽  
Signaling Activation

Abstract In acute myeloid leukemia (AML) standard therapies often induce complete remission, but patients frequently relapse and die of the disease. Leukemia stem cells (LSCs) have self-renewal potential and ability to recapitulate the disease. Our goal is to define the molecular mechanisms that allow AML to relapse. We have previously shown that activated NRAS (NRASG12V) facilitates self-renewal in the LSC-enriched subpopulation in a mouse model of AML (Mll-AF9/NRASG12V, Sachs et al. Blood 2014). We subsequently utilized single-cell RNA sequencing of the LSCs from this model to define and validate the only subset of the LSC-enriched population that can efficiently transplant leukemia in mice. We hypothesize that NRASG12V exerts a unique signaling profile that directs self-renewal in this subset of LSCs. Understanding these pathways at the single-cell level would enable us to design rational therapeutics that would prevent relapse in AML. We used mass cytometry (CyTOF2) to define the signaling activation state of LSC subsets in our AML model. Similar to flow cytometry, mass cytometry provides quantitative measurements of cell-surface and intracellular proteins at the single-cell level. In addition, it can simultaneously and accurately measure over 40 proteins, allowing us to quantitate a panel of intracellular signaling molecules in well-defined immunophenotypic leukemia subpopulations. We previously reported that the LSC-enriched population in this leukemia model is Mac1LowKit+Sca1+ (MKS) and subsequently showed that the self-renewing subset within the MKS population is MKSCD36LowCD69High. In contrast, the MKSCD36HighCD69Low population is incapable of transplanting leukemia in mice. The MKS cells displayed elevated levels of activated signaling molecules relative to the non-MKS population. Comparing the MKS subsets to each other, we found that the self-renewing MKSCD36LowCD69High population displayed significantly higher levels of several signaling molecules including Myc, NF-kB, and β-catenin relative to MKSCD36HighCD69Low cells (which lack self-renewal capacity). We reasoned that self-renewal might be mediated through these signaling molecules uniquely elevated in MKSCD36LowCD69High cells. Next, we sought to define the global signaling activation network within individual MKS subsets to determine if the signaling cascades and dependencies vary between these populations. We used Bayesian network modeling (Sachs K et al. Science 2005) to compare the statistical relationships between these signaling molecules, at the single-cell level. Signaling molecules that impact the levels of downstream effectors can be inferred using this approach. Using this method, we found that the signaling activation network does not significantly vary between MKS subsets. These observations suggest that self-renewal may be driven by alteration in the levels of signaling intermediates rather than alternate signal transduction architecture. We previously found that NRASG12V-mediated signals drive self-renewal in this AML model (Sachs Z. et al. Blood 2014). We used this model to ask which of these self-renewal-associated signaling molecules might be NRASG12V-regulated. We abolished NRASG12V transgene expression in these mice and harvested leukemia cells 72 hours later (per our standard lab protocol). Using this approach, found that self-renewal-associated signaling molecules, including NF-kB and β-catenin, are significantly reduced after NRASG12V-withdrawal indicating that NRASG12V -dependent signaling likely leads to the increase in these signaling molecules. In conclusion, we used mass cytometry analysis to identify the LSC self-renewal-associated signaling state in a murine model of AML and show that NRASG12V activates this signaling program. These data can be used to rationally design therapeutics such as small molecule inhibitors to target self-renewal-specific signaling and prevent relapse in AML. Disclosures No relevant conflicts of interest to declare.

Ability of Early Acting Cytokines to Directly Promote Survival and Suppress Apoptosis of Human Primitive CD34+CD38− Bone Marrow Cells With Multilineage Potential at the Single-Cell Level: Key Role of Thrombopoietin

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2282-2292 ◽  
Author(s):  
Ole J. Borge ◽  
Veslemøy Ramsfjell ◽  
Li Cui ◽  
Sten E.W. Jacobsen
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Single Cell ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
High Dose ◽  
Single Cell Level ◽  
Cell Level ◽  
Differentiation Potential

Purified primitive progenitor/stem cells from bone marrow represent likely target populations for ex vivo expansion of stem cells to be used in high-dose chemotherapy or gene therapy. Whereas such primitive progenitor cells require combined stimulation by multiple cytokines for growth, some cytokines selectively promote viability rather than growth when acting individually. We investigated here for the first time the direct effects of cytokines on survival of primitive CD34+CD38− human bone marrow progenitor cells at the single-cell level. Interleukin-3 (IL-3) and the ligands for c-kit (KL) and flt3 (FL) had direct and selective viability-promoting effects on a small fraction of CD34+CD38− but not CD34+CD38+ progenitor cells. Interestingly, the recently cloned thrombopoietin (Tpo), although stimulating little growth, kept most CD34+CD38− progenitors viable after prolonged culture, maintaining twofold and fourfold more progenitors viable than KL and IL-3, respectively. A high fraction of these progenitors had a combined myeloid and erythroid differentiation potential, as well as capacity for prolonged production of progenitor cells under stroma-independent conditions. In addition, Tpo promoted viability of CD34+CD38− long-term culture-initiating cells, further supporting the idea that Tpo promotes viability of primitive human progenitor cells. Finally, Tpo suppressed apoptosis of CD34+CD38− cells in culture. Thus, the present studies show a novel effect of Tpo, implicating a potential role of this cytokine in maintaining quiescent primitive human progenitor cells viable.

scholarly journals Ability of Early Acting Cytokines to Directly Promote Survival and Suppress Apoptosis of Human Primitive CD34+CD38− Bone Marrow Cells With Multilineage Potential at the Single-Cell Level: Key Role of Thrombopoietin

Blood ◽  
1997 ◽  
Vol 90 (6) ◽  
pp. 2282-2292 ◽  
Author(s):  
Ole J. Borge ◽  
Veslemøy Ramsfjell ◽  
Li Cui ◽  
Sten E.W. Jacobsen
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Single Cell ◽  
Progenitor Cells ◽  
Bone Marrow Cells ◽  
High Dose ◽  
Single Cell Level ◽  
Cell Level ◽  
Differentiation Potential

Abstract Purified primitive progenitor/stem cells from bone marrow represent likely target populations for ex vivo expansion of stem cells to be used in high-dose chemotherapy or gene therapy. Whereas such primitive progenitor cells require combined stimulation by multiple cytokines for growth, some cytokines selectively promote viability rather than growth when acting individually. We investigated here for the first time the direct effects of cytokines on survival of primitive CD34+CD38− human bone marrow progenitor cells at the single-cell level. Interleukin-3 (IL-3) and the ligands for c-kit (KL) and flt3 (FL) had direct and selective viability-promoting effects on a small fraction of CD34+CD38− but not CD34+CD38+ progenitor cells. Interestingly, the recently cloned thrombopoietin (Tpo), although stimulating little growth, kept most CD34+CD38− progenitors viable after prolonged culture, maintaining twofold and fourfold more progenitors viable than KL and IL-3, respectively. A high fraction of these progenitors had a combined myeloid and erythroid differentiation potential, as well as capacity for prolonged production of progenitor cells under stroma-independent conditions. In addition, Tpo promoted viability of CD34+CD38− long-term culture-initiating cells, further supporting the idea that Tpo promotes viability of primitive human progenitor cells. Finally, Tpo suppressed apoptosis of CD34+CD38− cells in culture. Thus, the present studies show a novel effect of Tpo, implicating a potential role of this cytokine in maintaining quiescent primitive human progenitor cells viable.

Metabolic Regulation and Related Molecular Mechanisms in Various Stem Cell Functions

2020 ◽  
Vol 15 (6) ◽  
pp. 531-546 ◽  
Author(s):  
Hwa-Yong Lee ◽  
In-Sun Hong
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Oxidative Phosphorylation ◽  
Metabolic Pathways ◽  
Critical Role ◽  
The Self ◽  
Specific Cell ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Cell Functions

Recent studies on the mechanisms that link metabolic changes with stem cell fate have deepened our understanding of how specific metabolic pathways can regulate various stem cell functions during the development of an organism. Although it was originally thought to be merely a consequence of the specific cell state, metabolism is currently known to play a critical role in regulating the self-renewal capacity, differentiation potential, and quiescence of stem cells. Many studies in recent years have revealed that metabolic pathways regulate various stem cell behaviors (e.g., selfrenewal, migration, and differentiation) by modulating energy production through glycolysis or oxidative phosphorylation and by regulating the generation of metabolites, which can modulate multiple signaling pathways. Therefore, a more comprehensive understanding of stem cell metabolism could allow us to establish optimal culture conditions and differentiation methods that would increase stem cell expansion and function for cell-based therapies. However, little is known about how metabolic pathways regulate various stem cell functions. In this context, we review the current advances in metabolic research that have revealed functional roles for mitochondrial oxidative phosphorylation, anaerobic glycolysis, and oxidative stress during the self-renewal, differentiation and aging of various adult stem cell types. These approaches could provide novel strategies for the development of metabolic or pharmacological therapies to promote the regenerative potential of stem cells and subsequently promote their therapeutic utility.

scholarly journals Single-Cell Transcriptome Analysis as a Promising Tool to Study Pluripotent Stem Cell Reprogramming

2021 ◽  
Vol 22 (11) ◽  
pp. 5988
Author(s):  
Hyun Kyu Kim ◽  
Tae Won Ha ◽  
Man Ryul Lee
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Cell Fate ◽  
Human Cell ◽  
Transcriptome Analysis ◽  
Single Cell Analysis ◽  
Embryonic Stem ◽  
Single Cell Level ◽  
Cell Analysis ◽  
Cell Level

Cells are the basic units of all organisms and are involved in all vital activities, such as proliferation, differentiation, senescence, and apoptosis. A human body consists of more than 30 trillion cells generated through repeated division and differentiation from a single-cell fertilized egg in a highly organized programmatic fashion. Since the recent formation of the Human Cell Atlas consortium, establishing the Human Cell Atlas at the single-cell level has been an ongoing activity with the goal of understanding the mechanisms underlying diseases and vital cellular activities at the level of the single cell. In particular, transcriptome analysis of embryonic stem cells at the single-cell level is of great importance, as these cells are responsible for determining cell fate. Here, we review single-cell analysis techniques that have been actively used in recent years, introduce the single-cell analysis studies currently in progress in pluripotent stem cells and reprogramming, and forecast future studies.

scholarly journals High Throughput Immunophenotyping and Expression Profiling at Single Cell Level Reveal BCR-ABL1 Dependent Surface Markers of Chronic Myeloid Leukemia Stem Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2920-2920
Author(s):  
Marianna Romzova ◽  
Dagmar Smitalova ◽  
Peter Taus ◽  
Jiri Mayer ◽  
Martin Culen
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Expression Profiling ◽  
Myeloid Leukemia ◽  
Single Cells ◽  
Transcript Level ◽  
Surface Expression ◽  
Surface Markers ◽  
Cell Level ◽  
Tki Treatment

BACKGROUND: Bcr-abl1 oncogene targeted treatment with tyrosine kinase inhibitors (TKI) showed an impressive efficacy against proliferating chronic myeloid leukemia (CML) cells. However, rapid relapses in more than half of CML patients after discontinuation of the treatment suggest a presence of quiescent leukemic stem cells inherently resistant to BCR-ABL1 inhibition. Understanding the heterogeneity of CML stem cell compartment is crucial for preventing the treatment failure. Specificity of already established leukemic stem cell (LSC) markers has been tested mainly in bulk CD34+CD38- populations at diagnosis. Phenotypes and molecular signatures of therapy resistant BCR ABL1 positive stem cells is however yet to be established. AIMS: Identification of BCR-ABL1 dependent LSC markers at single cell level by direct comparison their surface and transcript expression with the levels and the presence of BCR-ABL1 transcript at diagnosis and after administration of TKI treatment. METHODS: Total number of 375 cells were obtained from bone marrow and peripheral blood of 4 chronic phase CML patients. Cells were collected prior any treatment and three months after TKI treatment initiation. Normal bone marrow cells and BCR-ABL1 positive K562 cell line were used as controls. Indexed immuno-phenotyping and sorting of CD34+CD38- single cells was performed using a panel of 11 specific surface markers. Collected single cells were lysed and cDNA was enriched for 11 targets using 22 cycle pre-amplification. Expression profiling was carried on SmartChip real-time PCR system (Takara Bio) detecting following genes: BCR-ABL1, CD26, CD25, IL1-Rap, CD56, CD90, CD93, CD69, KI67, and control genes GUS and HPRT. Unsupervised clustering was performed using principal component analysis (PCA). Correlations were measured by Spearman rank method. RESULTS: At diagnosis, majority of BCR-ABL1+ C34+CD38- stem cells co-express IL1-Rap, CD26, and CD69 on their surface (88%, 82%, 78% overlap). Only 56% of BCR-ABL1+ cells positive for aforementioned markers co-express CD25, 28% CD93 and 16% CD56. The expression of these markers could also be detected in 4-11% of BCR-ABL1- cell, although this could be technical inaccuracy caused by the single cell profiling. CD90 marker did not show any correlation with BCR-ABL1 expression. At transcript level the expression of IL-1Rap, CD26, CD25 and CD56 was observed in 62%, 52% 45% and 16% BCR-ABL1+ cells, and up to 7% of BCR-ABL1- cells. CD69 expression was observed in 90% of BCR-ABL+ cells at transcript level, but also in 71% BCR-ABL- cells. BCR-ABL1 independent expression was observed for cKIT. (60% vs. 76 % in positive vs negative). Finally proliferation marker KI67 was expressed only in 6% of the BCR-ABL1+ cells. PCA analysis divided cells into several distinct clusters with BCR-ABL1 as the main contributor, and cKIT, CD69 and CD26, IL-1RAP as other significant factors. Interestingly BCR-ABL1+ cells collected during TKI treatment showed persistent surface expression of IL-1Rap and CD26, while CD56, CD69 and CD93 were only on part of the BCR-ABL1+ cells. CD25 was significantly deregulated during TKI treatment. CONCLUSION: At diagnosis up to 80% of LSC co-express 3 specific surface markers - IL-1RAP, CD26 and CD69. Variable portion of LSC co-express additional markers such are CD25, CD56 and CD93. During TKI treatment the surface expression of majority of markers is decreased, where the best correlated LSC marker is IL-1Rap, followed by CD26 and CD69. CD56 marker seems to persist in the same proportion of cells while CD25 disappears. cKIT is highly expressed in normal BM and HSC from CML patients, but also in some LSC. CD34+CD38- cells show non-proliferating phenotype. Disclosures Mayer: AOP Orphan Pharmaceuticals AG: Research Funding.

scholarly journals Microfluidic Platforms Designed for Morphological and Photosynthetic Investigations of Chlamydomonas reinhardtii on a Single-Cell Level

Cells ◽  
2022 ◽  
Vol 11 (2) ◽  
pp. 285
Author(s):  
Eszter Széles ◽  
Krisztina Nagy ◽  
Ágnes Ábrahám ◽  
Sándor Kovács ◽  
Anna Podmaniczki ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Single Cell ◽  
Single Cells ◽  
Fluorescence Induction ◽  
Photochemical Quenching ◽  
Support Surface ◽  
Single Cell Level ◽  
Microfluidic Platforms ◽  
Cell Level ◽  
Non Photochemical Quenching

Chlamydomonas reinhardtii is a model organism of increasing biotechnological importance, yet, the evaluation of its life cycle processes and photosynthesis on a single-cell level is largely unresolved. To facilitate the study of the relationship between morphology and photochemistry, we established microfluidics in combination with chlorophyll a fluorescence induction measurements. We developed two types of microfluidic platforms for single-cell investigations: (i) The traps of the “Tulip” device are suitable for capturing and immobilizing single cells, enabling the assessment of their photosynthesis for several hours without binding to a solid support surface. Using this “Tulip” platform, we performed high-quality non-photochemical quenching measurements and confirmed our earlier results on bulk cultures that non-photochemical quenching is higher in ascorbate-deficient mutants (Crvtc2-1) than in the wild-type. (ii) The traps of the “Pot” device were designed for capturing single cells and allowing the growth of the daughter cells within the traps. Using our most performant “Pot” device, we could demonstrate that the FV/FM parameter, an indicator of photosynthetic efficiency, varies considerably during the cell cycle. Our microfluidic devices, therefore, represent versatile platforms for the simultaneous morphological and photosynthetic investigations of C. reinhardtii on a single-cell level.

scholarly journals A single-cell Raman-based platform to identify developmental stages of human pluripotent stem cell-derived neurons

2020 ◽  
Vol 117 (31) ◽  
pp. 18412-18423 ◽  
Author(s):  
Chia-Chen Hsu ◽  
Jiabao Xu ◽  
Bas Brinkhof ◽  
Hui Wang ◽  
Zhanfeng Cui ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Single Cell ◽  
Raman Spectra ◽  
Large Scale ◽  
Developmental Stages ◽  
Single Cells ◽  
Classification Model ◽  
Label Free ◽  
Machine Learning Classification

Stem cells with the capability to self-renew and differentiate into multiple cell derivatives provide platforms for drug screening and promising treatment options for a wide variety of neural diseases. Nevertheless, clinical applications of stem cells have been hindered partly owing to a lack of standardized techniques to characterize cell molecular profiles noninvasively and comprehensively. Here, we demonstrate that a label-free and noninvasive single-cell Raman microspectroscopy (SCRM) platform was able to identify neural cell lineages derived from clinically relevant human induced pluripotent stem cells (hiPSCs). By analyzing the intrinsic biochemical profiles of single cells at a large scale (8,774 Raman spectra in total), iPSCs and iPSC-derived neural cells can be distinguished by their intrinsic phenotypic Raman spectra. We identified a Raman biomarker from glycogen to distinguish iPSCs from their neural derivatives, and the result was verified by the conventional glycogen detection assays. Further analysis with a machine learning classification model, utilizing t-distributed stochastic neighbor embedding (t-SNE)-enhanced ensemble stacking, clearly categorized hiPSCs in different developmental stages with 97.5% accuracy. The present study demonstrates the capability of the SCRM-based platform to monitor cell development using high content screening with a noninvasive and label-free approach. This platform as well as our identified biomarker could be extensible to other cell types and can potentially have a high impact on neural stem cell therapy.

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