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Nature Mar 2023Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell...
Haematopoietic stem cells (HSCs) are a rare cell type that reconstitute the entire blood and immune systems after transplantation and can be used as a curative cell therapy for a variety of haematological diseases. However, the low number of HSCs in the body makes both biological analyses and clinical application difficult, and the limited extent to which human HSCs can be expanded ex vivo remains a substantial barrier to the wider and safer therapeutic use of HSC transplantation. Although various reagents have been tested in attempts to stimulate the expansion of human HSCs, cytokines have long been thought to be essential for supporting HSCs ex vivo. Here we report the establishment of a culture system that allows the long-term ex vivo expansion of human HSCs, achieved through the complete replacement of exogenous cytokines and albumin with chemical agonists and a caprolactam-based polymer. A phosphoinositide 3-kinase activator, in combination with a thrombopoietin-receptor agonist and the pyrimidoindole derivative UM171, were sufficient to stimulate the expansion of umbilical cord blood HSCs that are capable of serial engraftment in xenotransplantation assays. Ex vivo HSC expansion was further supported by split-clone transplantation assays and single-cell RNA-sequencing analysis. Our chemically defined expansion culture system will help to advance clinical HSC therapies.
Topics: Humans; Cell Proliferation; Clone Cells; Cytokines; Fetal Blood; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Phosphatidylinositol 3-Kinases; Cell Culture Techniques; Albumins; Caprolactam; Polymers; Receptors, Thrombopoietin; Transplantation, Heterologous; Single-Cell Gene Expression Analysis
PubMed: 36813966
DOI: 10.1038/s41586-023-05739-9 -
Nature Aug 2023Even among genetically identical cancer cells, resistance to therapy frequently emerges from a small subset of those cells. Molecular differences in rare individual...
Even among genetically identical cancer cells, resistance to therapy frequently emerges from a small subset of those cells. Molecular differences in rare individual cells in the initial population enable certain cells to become resistant to therapy; however, comparatively little is known about the variability in the resistance outcomes. Here we develop and apply FateMap, a framework that combines DNA barcoding with single-cell RNA sequencing, to reveal the fates of hundreds of thousands of clones exposed to anti-cancer therapies. We show that resistant clones emerging from single-cell-derived cancer cells adopt molecularly, morphologically and functionally distinct resistant types. These resistant types are largely predetermined by molecular differences between cells before drug addition and not by extrinsic factors. Changes in the dose and type of drug can switch the resistant type of an initial cell, resulting in the generation and elimination of certain resistant types. Samples from patients show evidence for the existence of these resistant types in a clinical context. We observed diversity in resistant types across several single-cell-derived cancer cell lines and cell types treated with a variety of drugs. The diversity of resistant types as a result of the variability in intrinsic cell states may be a generic feature of responses to external cues.
Topics: Humans; Clone Cells; DNA Barcoding, Taxonomic; Drug Resistance, Neoplasm; Neoplasms; RNA-Seq; Single-Cell Gene Expression Analysis; Tumor Cells, Cultured; Antineoplastic Agents
PubMed: 37468627
DOI: 10.1038/s41586-023-06342-8 -
Nature Jul 2019Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation, which is a curative therapy for numerous diseases...
Multipotent self-renewing haematopoietic stem cells (HSCs) regenerate the adult blood system after transplantation, which is a curative therapy for numerous diseases including immunodeficiencies and leukaemias. Although substantial effort has been applied to identifying HSC maintenance factors through the characterization of the in vivo bone-marrow HSC microenvironment or niche, stable ex vivo HSC expansion has previously been unattainable. Here we describe the development of a defined, albumin-free culture system that supports the long-term ex vivo expansion of functional mouse HSCs. We used a systematic optimization approach, and found that high levels of thrombopoietin synergize with low levels of stem-cell factor and fibronectin to sustain HSC self-renewal. Serum albumin has long been recognized as a major source of biological contaminants in HSC cultures; we identify polyvinyl alcohol as a functionally superior replacement for serum albumin that is compatible with good manufacturing practice. These conditions afford between 236- and 899-fold expansions of functional HSCs over 1 month, although analysis of clonally derived cultures suggests that there is considerable heterogeneity in the self-renewal capacity of HSCs ex vivo. Using this system, HSC cultures that are derived from only 50 cells robustly engraft in recipient mice without the normal requirement for toxic pre-conditioning (for example, radiation), which may be relevant for HSC transplantation in humans. These findings therefore have important implications for both basic HSC research and clinical haematology.
Topics: Animals; Cell Culture Techniques; Cell Proliferation; Cell Self Renewal; Clone Cells; Culture Media; Female; Fibronectins; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Male; Mice; Polyvinyl Alcohol; Serum Albumin; Stem Cell Factor; Thrombopoietin; Time Factors; Transplantation Conditioning
PubMed: 31142833
DOI: 10.1038/s41586-019-1244-x -
Nature Aug 2021Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure, where some cancer cells can enter a reversible drug-tolerant persister state...
Non-genetic mechanisms have recently emerged as important drivers of cancer therapy failure, where some cancer cells can enter a reversible drug-tolerant persister state in response to treatment. Although most cancer persisters remain arrested in the presence of the drug, a rare subset can re-enter the cell cycle under constitutive drug treatment. Little is known about the non-genetic mechanisms that enable cancer persisters to maintain proliferative capacity in the presence of drugs. To study this rare, transiently resistant, proliferative persister population, we developed Watermelon, a high-complexity expressed barcode lentiviral library for simultaneous tracing of each cell's clonal origin and proliferative and transcriptional states. Here we show that cycling and non-cycling persisters arise from different cell lineages with distinct transcriptional and metabolic programs. Upregulation of antioxidant gene programs and a metabolic shift to fatty acid oxidation are associated with persister proliferative capacity across multiple cancer types. Impeding oxidative stress or metabolic reprogramming alters the fraction of cycling persisters. In human tumours, programs associated with cycling persisters are induced in minimal residual disease in response to multiple targeted therapies. The Watermelon system enabled the identification of rare persister lineages that are preferentially poised to proliferate under drug pressure, thus exposing new vulnerabilities that can be targeted to delay or even prevent disease recurrence.
Topics: Antioxidants; Cell Cycle; Cell Line, Tumor; Cell Lineage; Cell Proliferation; Cell Survival; Clone Cells; DNA Barcoding, Taxonomic; Fatty Acids; Gene Expression Regulation, Neoplastic; Humans; Lentivirus; Neoplasm Recurrence, Local; Neoplasms; Oncogene Proteins; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Transcription, Genetic
PubMed: 34381210
DOI: 10.1038/s41586-021-03796-6 -
Nature Mar 2024The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived haematopoietic stem cells (HSCs)....
The human blood system is maintained through the differentiation and massive amplification of a limited number of long-lived haematopoietic stem cells (HSCs). Perturbations to this process underlie diverse diseases, but the clonal contributions to human haematopoiesis and how this changes with age remain incompletely understood. Although recent insights have emerged from barcoding studies in model systems, simultaneous detection of cell states and phylogenies from natural barcodes in humans remains challenging. Here we introduce an improved, single-cell lineage-tracing system based on deep detection of naturally occurring mitochondrial DNA mutations with simultaneous readout of transcriptional states and chromatin accessibility. We use this system to define the clonal architecture of HSCs and map the physiological state and output of clones. We uncover functional heterogeneity in HSC clones, which is stable over months and manifests as both differences in total HSC output and biases towards the production of different mature cell types. We also find that the diversity of HSC clones decreases markedly with age, leading to an oligoclonal structure with multiple distinct clonal expansions. Our study thus provides a clonally resolved and cell-state-aware atlas of human haematopoiesis at single-cell resolution, showing an unappreciated functional diversity of human HSC clones and, more broadly, paving the way for refined studies of clonal dynamics across a range of tissues in human health and disease.
Topics: Humans; Cell Lineage; Chromatin; Clone Cells; DNA, Mitochondrial; Hematopoiesis; Hematopoietic Stem Cells; Mutation; Single-Cell Analysis; Transcription, Genetic; Aging
PubMed: 38253266
DOI: 10.1038/s41586-024-07066-z -
Nature Dec 2018Direct lineage reprogramming involves the conversion of cellular identity. Single-cell technologies are useful for deconstructing the considerable heterogeneity that...
Direct lineage reprogramming involves the conversion of cellular identity. Single-cell technologies are useful for deconstructing the considerable heterogeneity that emerges during lineage conversion. However, lineage relationships are typically lost during cell processing, complicating trajectory reconstruction. Here we present 'CellTagging', a combinatorial cell-indexing methodology that enables parallel capture of clonal history and cell identity, in which sequential rounds of cell labelling enable the construction of multi-level lineage trees. CellTagging and longitudinal tracking of fibroblast to induced endoderm progenitor reprogramming reveals two distinct trajectories: one leading to successfully reprogrammed cells, and one leading to a 'dead-end' state, paths determined in the earliest stages of lineage conversion. We find that expression of a putative methyltransferase, Mettl7a1, is associated with the successful reprogramming trajectory; adding Mettl7a1 to the reprogramming cocktail increases the yield of induced endoderm progenitors. Together, these results demonstrate the utility of our lineage-tracing method for revealing the dynamics of direct reprogramming.
Topics: Animals; Cell Lineage; Cell Separation; Cell Tracking; Cellular Reprogramming; Clone Cells; Endoderm; Fibroblasts; HEK293 Cells; Humans; Methyltransferases; Mice; Single-Cell Analysis; Stem Cells; Time Factors
PubMed: 30518857
DOI: 10.1038/s41586-018-0744-4 -
Nature Aug 2018Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional...
Human cancer cell lines are the workhorse of cancer research. Although cell lines are known to evolve in culture, the extent of the resultant genetic and transcriptional heterogeneity and its functional consequences remain understudied. Here we use genomic analyses of 106 human cell lines grown in two laboratories to show extensive clonal diversity. Further comprehensive genomic characterization of 27 strains of the common breast cancer cell line MCF7 uncovered rapid genetic diversification. Similar results were obtained with multiple strains of 13 additional cell lines. Notably, genetic changes were associated with differential activation of gene expression programs and marked differences in cell morphology and proliferation. Barcoding experiments showed that cell line evolution occurs as a result of positive clonal selection that is highly sensitive to culture conditions. Analyses of single-cell-derived clones demonstrated that continuous instability quickly translates into heterogeneity of the cell line. When the 27 MCF7 strains were tested against 321 anti-cancer compounds, we uncovered considerably different drug responses: at least 75% of compounds that strongly inhibited some strains were completely inactive in others. This study documents the extent, origins and consequences of genetic variation within cell lines, and provides a framework for researchers to measure such variation in efforts to support maximally reproducible cancer research.
Topics: Breast Neoplasms; Cell Proliferation; Cell Shape; Clone Cells; Evolution, Molecular; Genetic Variation; Genomic Instability; Humans; MCF-7 Cells; Reproducibility of Results; Transcription, Genetic
PubMed: 30089904
DOI: 10.1038/s41586-018-0409-3 -
Nature Jan 2022The state and behaviour of a cell can be influenced by both genetic and environmental factors. In particular, tumour progression is determined by underlying genetic...
The state and behaviour of a cell can be influenced by both genetic and environmental factors. In particular, tumour progression is determined by underlying genetic aberrations as well as the makeup of the tumour microenvironment. Quantifying the contributions of these factors requires new technologies that can accurately measure the spatial location of genomic sequence together with phenotypic readouts. Here we developed slide-DNA-seq, a method for capturing spatially resolved DNA sequences from intact tissue sections. We demonstrate that this method accurately preserves local tumour architecture and enables the de novo discovery of distinct tumour clones and their copy number alterations. We then apply slide-DNA-seq to a mouse model of metastasis and a primary human cancer, revealing that clonal populations are confined to distinct spatial regions. Moreover, through integration with spatial transcriptomics, we uncover distinct sets of genes that are associated with clone-specific genetic aberrations, the local tumour microenvironment, or both. Together, this multi-modal spatial genomics approach provides a versatile platform for quantifying how cell-intrinsic and cell-extrinsic factors contribute to gene expression, protein abundance and other cellular phenotypes.
Topics: Animals; Clone Cells; Colorectal Neoplasms; DNA Copy Number Variations; Genomics; Humans; Mice; Phenotype; RNA-Seq; Sequence Analysis, DNA; Transcription, Genetic; Transcriptome
PubMed: 34912115
DOI: 10.1038/s41586-021-04217-4 -
Nature Nov 2020Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk...
Myeloid malignancies, including acute myeloid leukaemia (AML), arise from the expansion of haematopoietic stem and progenitor cells that acquire somatic mutations. Bulk molecular profiling has suggested that mutations are acquired in a stepwise fashion: mutant genes with high variant allele frequencies appear early in leukaemogenesis, and mutations with lower variant allele frequencies are thought to be acquired later. Although bulk sequencing can provide information about leukaemia biology and prognosis, it cannot distinguish which mutations occur in the same clone(s), accurately measure clonal complexity, or definitively elucidate the order of mutations. To delineate the clonal framework of myeloid malignancies, we performed single-cell mutational profiling on 146 samples from 123 patients. Here we show that AML is dominated by a small number of clones, which frequently harbour co-occurring mutations in epigenetic regulators. Conversely, mutations in signalling genes often occur more than once in distinct subclones, consistent with increasing clonal diversity. We mapped clonal trajectories for each sample and uncovered combinations of mutations that synergized to promote clonal expansion and dominance. Finally, we combined protein expression with mutational analysis to map somatic genotype and clonal architecture with immunophenotype. Our findings provide insights into the pathogenesis of myeloid transformation and how clonal complexity evolves with disease progression.
Topics: Cell Separation; Clone Cells; DNA Mutational Analysis; Humans; Immunophenotyping; Mutation; Myeloproliferative Disorders; Single-Cell Analysis
PubMed: 33116311
DOI: 10.1038/s41586-020-2864-x -
Science (New York, N.Y.) May 2011Pluripotent cells in the embryo can generate all cell types, but lineage-restricted cells are generally thought to replenish adult tissues. Planarians are flatworms and...
Pluripotent cells in the embryo can generate all cell types, but lineage-restricted cells are generally thought to replenish adult tissues. Planarians are flatworms and regenerate from tiny body fragments, a process requiring a population of proliferating cells (neoblasts). Whether regeneration is accomplished by pluripotent cells or by the collective activity of multiple lineage-restricted cell types is unknown. We used ionizing radiation and single-cell transplantation to identify neoblasts that can form large descendant-cell colonies in vivo. These clonogenic neoblasts (cNeoblasts) produce cells that differentiate into neuronal, intestinal, and other known postmitotic cell types and are distributed throughout the body. Single transplanted cNeoblasts restored regeneration in lethally irradiated hosts. We conclude that broadly distributed, adult pluripotent stem cells underlie the remarkable regenerative abilities of planarians.
Topics: Adult Stem Cells; Animals; Base Sequence; Cell Differentiation; Cell Lineage; Cell Proliferation; Cell Separation; Clone Cells; Genes, Helminth; Genotype; Intestines; Molecular Sequence Data; Neurons; Planarians; Pluripotent Stem Cells; Regeneration
PubMed: 21566185
DOI: 10.1126/science.1203983