scholarly journals Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment

PLoS Biology ◽  
2017 ◽  
Vol 15 (7) ◽  
pp. e2001867 ◽  
Author(s):  
Alice Moussy ◽  
Jérémie Cosette ◽  
Romuald Parmentier ◽  
Cindy da Silva ◽  
Guillaume Corre ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Time Lapse ◽  
Hematopoietic Cell ◽  
Dynamic Nature

scholarly journals Integrated time-lapse and single-cell transcription studies highlight the variable and dynamic nature of human hematopoietic cell fate commitment

2017 ◽  
Author(s):  
Alice Moussy ◽  
Jérémie Cosette ◽  
Romuald Parmentier ◽  
Cindy da Silva ◽  
Guillaume Corre ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Cycle ◽  
Single Cell ◽  
Cell Fate ◽  
Morphological Changes ◽  
Gene Expression Pattern ◽  
Time Lapse ◽  
Dynamic Nature ◽  
Transcriptional Changes ◽  
Fate Decision

AbstractIndividual cells take lineage commitment decisions in a way that is not necessarily uniform. We address this issue by characterizing transcriptional changes in cord blood derived CD34+ cells at the single-cell level and integrating data with cell division history and morphological changes determined by time-lapse microscopy. We show, that major transcriptional changes leading to a multilineage-primed gene expression state occur very rapidly during the first cell cycle. One of the two stable lineage-primed patterns emerges gradually in each cell with variable timing. Some cells reach a stable morphology and molecular phenotype by the end of the first cell cycle and transmit it clonally. Others fluctuate between the two phenotypes over several cell cycles. Our analysis highlights the dynamic nature and variable timing of cell fate commitment in hematopoietic cells, links the gene expression pattern to cell morphology and identifies a new category of cells with fluctuating phenotypic characteristics, demonstrating the complexity of the fate decision process, away from a simple binary switch between two options as it is usually envisioned.

scholarly journals Label free, quantitative single-cell fate tracking of time-lapse movies

MethodsX ◽  
2019 ◽  
Vol 6 ◽  
pp. 2468-2475 ◽  
Author(s):  
C. Elizabeth Caldon ◽  
Andrew Burgess
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Time Lapse ◽  
Label Free ◽  
Cell Fate Tracking

scholarly journals The Single-Cell Transcriptional Landscape of Human Hematopoietic Progenitors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1266-1266
Author(s):  
Mariana Loperfido ◽  
Danilo Pellin ◽  
Cristina Baricordi ◽  
Samuel Louis Wolock ◽  
Allon Moshe Klein ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dendritic Cell ◽  
Mast Cells ◽  
Single Cell ◽  
Cell Fate ◽  
Blood Cells ◽  
Single Cells ◽  
Hematopoietic Cell ◽  
Hematopoietic Progenitors ◽  
Cd34 Cells

Abstract In humans, hematopoietic stem/progenitor cells (HSPCs) constitute a heterogeneous population in the bone marrow (BM) endowed with the potential of generating and maintaining an adequate and diverse pool of blood cells lifelong. However, the nature of hematopoietic cell fate choice remains unresolved and is still highly contentious. In fact, despite the recent efforts and the advent of single-cell technologies, conflicting classical, early-split or continuous models for the generation of human blood cells still coexist in the literature as of today. We here used single-cell RNA-Seq (scRNA-Seq) to provide a detailed map of the transcriptional states of hematopoietic progenitors in the human BM. We believe that this work provides unprecedented insights into the structure of early cell-fate choices of the human hematopoietic system. Single-cell analysis of human HSPCs is a well-worn path, but all previous studies aimed at investigating the shape of early cell fate choices human hematopoiesis have been based on the use of immune-selected CD34+ cells. We too started by separating CD34+ cells purified by magnetic beads selection into seven subpopulations, marking cells of differing fate potential and we successfully tagged and sequenced the transcriptome of 6,011 single cells. Our scRNA-Seq map of CD34+ subpopulations shows that HSPCs do not undergo a single-step transition from HSPCs to unilineage states. Instead, they form a clear hierarchy. The earliest fate split separates erythroid-megakaryocyte progenitors from lymphoid-myeloid progenitors, which separate further into lymphoid, dendritic cell and granulocytic progenitors. We noted, however, that branches towards basophils/eosinophils/mast cells and monocytes commitments were missing. Indeed, many cells negative for mature lineage markers in human BM are CD34low/- and could account for additional transitional states at which the CD34 expression is rapidly downregulated, therefore greatly reducing their probability of capture. Thus, differently from previous works, we also extended our analysis to the whole bone marrow fraction lacking the main markers of terminal differentiation (Lin- cells). In doing so we used a novel graded FACS-sorting strategy that corrects for expansion of cells as they differentiated, and thus allows examining early states alongside later ones that comprise the vast majority of Lin- progenitors. In fractionating the cells by maturity, we made use of a cell surface marker, GeneA, which we identified from the initial data set as capturing cells that are multipotent until just beyond the first branch-point.The result of this analysis was a high resolution landscape from the transcriptome of 15,401 single cells, showing multiple branch points, and a topology that remarkably similar to what we reported in mice (Tusi et al. Nature 2018). This map defines the early branching of dendritic cell vs monocyte precursors and supports the novel concept that basophils/eosinophils/mast cells do not derive from common granulocytes/monocytes progenitors but originate instead from what are currently defined as megakaryo/erythroid progenitors.By the in depth analysis of our single-cell maps we generated a number of information of value for the broader hematology community: * We put the "classical" CD34+ FACS-based cell subpopulations into the context of the newly generated BM Lin- population structure (valuable for transplantation research) * We investigated the expression of transcription factors along differentiation trajectories and branching points (valuable for in vitro reprogramming efforts) * We analyzed the expression of several blood cancer genes over the main differentiation hubs and hematopoietic branches (valuable for the study, diagnosis and therapy of hematological tumors) * We provided a detailed comparison of the expression of gene orthologues in human vs mouse hematopoiesis (valuable for the modeling of blood cell disorders in the mouse) * We identified and validated GeneA as a reliable marker for the earliest branches of HSPCs specification in transplantation cell products (valuable for transplantation research and gene therapy) Overall, our study provides a detailed view of the early hematopoietic cell state hierarchy in humans and a valuable resource for HSPC biology. Disclosures No relevant conflicts of interest to declare.

scholarly journals Multiplexed live visualization of cell fate dynamics in hPSCs at single-cell resolution

2021 ◽  
Author(s):  
Sungmin Kim ◽  
Edward Ren ◽  
Paola Marco Casanova ◽  
Eugenia Piddini ◽  
Rafael Carazo Salas
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Fluorescent Proteins ◽  
Single Cells ◽  
Spatial Location ◽  
Time Lapse ◽  
Strongly Coupled ◽  
Pluripotency Marker ◽  
Tightly Coupled ◽  
Differentiation Marker

ABSTRACTLive imaging can provide powerful insights into developmental and cellular processes but availability of multiplexable reporters has been limiting. Here we describe ORACLE, a cell fate reporter class in which fluorescent proteins fused with the nucleoporin POM121 are driven by promoters of transcription factors of interest. ORACLE’s nuclear rim localisation therefore enables multiplexing with conventional nuclear reporters. We applied ORACLE to investigate the dynamics of pluripotency exit at single-cell level, using human pluripotent stem cells (hPSCs) imaged by multi-day time-lapse high-content microscopy. Using an ORACLE-OCT4 pluripotency marker we reveal that G1 phase length and OCT4 level are strongly coupled and that spatial location in a colony impacts the timing of pluripotency exit. Combining ORACLE-OCT4 and an ORACLE-SOX1 early neuronal differentiation marker, we visualize in real-time the dynamics of cell fate transition between pluripotency and early neural fate, and show that pluripotency exit and differentiation onset are likely not tightly coupled in single-cells. Thus ORACLE is a powerful tool to enable quantitative studies of spatiotemporal cell fate control.

scholarly journals Clonal analysis and dynamic imaging identify multipotency of individual Gallus gallus caudal hindbrain neural crest cells toward cardiac and enteric fates

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Weiyi Tang ◽  
Yuwei Li ◽  
Ang Li ◽  
Marianne E. Bronner
Keyword(s):  
Neural Crest ◽  
Single Cell ◽  
Cell Fate ◽  
Neural Crest Cells ◽  
Time Lapse ◽  
Cell Types ◽  
Clonal Analysis ◽  
Dynamic Imaging ◽  
Lineage Tracing ◽  
Enteric Ganglia

AbstractNeural crest stem cells arising from caudal hindbrain (often called cardiac and posterior vagal neural crest) migrate long distances to form cell types as diverse as heart muscle and enteric ganglia, abnormalities of which lead to common congenital birth defects. Here, we explore whether individual caudal hindbrain neural crest precursors are multipotent or predetermined toward these particular fates and destinations. To this end, we perform lineage tracing of chick neural crest cells at single-cell resolution using two complementary approaches: retrovirally mediated multiplex clonal analysis and single-cell photoconversion. Both methods show that the majority of these neural crest precursors are multipotent with many clones producing mesenchymal as well as neuronal derivatives. Time-lapse imaging demonstrates that sister cells can migrate in distinct directions, suggesting stochasticity in choice of migration path. Perturbation experiments further identify guidance cues acting on cells in the pharyngeal junction that can influence this choice; loss ofCXCR4signaling results in failure to migrate to the heart but no influence on migration toward the foregut, whereas loss ofRETsignaling does the opposite. Taken together, the results suggest that environmental influences rather than intrinsic information govern cell fate choice of multipotent caudal hindbrain neural crest cells.

scholarly journals A role for the fusogen eff-1 in epidermal stem cell number robustness in Caenorhabditis elegans

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sneha L. Koneru ◽  
Fu Xiang Quah ◽  
Ritobrata Ghose ◽  
Mark Hintze ◽  
Nicola Gritti ◽  
...  
Keyword(s):  
Stem Cell ◽  
Caenorhabditis Elegans ◽  
Cell Fate ◽  
Single Molecule ◽  
Phenotypic Variability ◽  
Low Frequency ◽  
Time Lapse ◽  
Cell Number ◽  
Seam Cell ◽  
Neuronal Tissues

AbstractDevelopmental patterning in Caenorhabditis elegans is known to proceed in a highly stereotypical manner, which raises the question of how developmental robustness is achieved despite the inevitable stochastic noise. We focus here on a population of epidermal cells, the seam cells, which show stem cell-like behaviour and divide symmetrically and asymmetrically over post-embryonic development to generate epidermal and neuronal tissues. We have conducted a mutagenesis screen to identify mutants that introduce phenotypic variability in the normally invariant seam cell population. We report here that a null mutation in the fusogen eff-1 increases seam cell number variability. Using time-lapse microscopy and single molecule fluorescence hybridisation, we find that seam cell division and differentiation patterns are mostly unperturbed in eff-1 mutants, indicating that cell fusion is uncoupled from the cell differentiation programme. Nevertheless, seam cell losses due to the inappropriate differentiation of both daughter cells following division, as well as seam cell gains through symmetric divisions towards the seam cell fate were observed at low frequency. We show that these stochastic errors likely arise through accumulation of defects interrupting the continuity of the seam and changing seam cell shape, highlighting the role of tissue homeostasis in suppressing phenotypic variability during development.

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