Single-cell technologies are transforming biomedical research, including the recent demonstration that unspliced pre-mRNA present in single-cell RNA-Seq permits prediction of future expression ...states. Here we apply this RNA velocity concept to an extended timecourse dataset covering mouse gastrulation and early organogenesis.
Intriguingly, RNA velocity correctly identifies epiblast cells as the starting point, but several trajectory predictions at later stages are inconsistent with both real-time ordering and existing knowledge. The most striking discrepancy concerns red blood cell maturation, with velocity-inferred trajectories opposing the true differentiation path. Investigating the underlying causes reveals a group of genes with a coordinated step-change in transcription, thus violating the assumptions behind current velocity analysis suites, which do not accommodate time-dependent changes in expression dynamics. Using scRNA-Seq analysis of chimeric mouse embryos lacking the major erythroid regulator Gata1, we show that genes with the step-changes in expression dynamics during erythroid differentiation fail to be upregulated in the mutant cells, thus underscoring the coordination of modulating transcription rate along a differentiation trajectory. In addition to the expected block in erythroid maturation, the Gata1-chimera dataset reveals induction of PU.1 and expansion of megakaryocyte progenitors. Finally, we show that erythropoiesis in human fetal liver is similarly characterized by a coordinated step-change in gene expression.
By identifying a limitation of the current velocity framework coupled with in vivo analysis of mutant cells, we reveal a coordinated step-change in gene expression kinetics during erythropoiesis, with likely implications for many other differentiation processes.
Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved > 200‐fold expansion of functional ...HSCs, but their molecular characterization has not been possible since the majority of cells are non‐HSCs and single cell‐initiated cultures have substantial clone‐to‐clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non‐HSCs in expansion cultures. By directly linking single‐clone functional transplantation data with single‐clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This “repopulation signature” (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.
Synopsis
This study presents a method for prospectively isolating HSCs from in vitro cultures. Paired molecular and functional analyses on a large number of individual clones identifies a gene expression signature that can identify functional HSCs from multiple cellular states.
EPCR, ESAM and Fgd5 permit prospective identification of functional HSCs from in vitro clones.
Molecular profiling of purified expanded HSCs shows that their profile is similar to proliferating fetal HSCs.
A specific gene signature identifies repopulating HSCs from a wide range of different cellular states, including human HSCs.
This study presents a method for prospectively isolating HSCs from in vitro cultures. Paired molecular and functional analyses on a large number of individual clones identifies a gene expression signature that can identify functional HSCs from multiple cellular states.
Haematopoietic stem cells (HSCs) are widely studied by HSC transplantation into immune- and blood-cell-depleted recipients. Single HSCs can rebuild the system after transplantation. Chromosomal ...marking, viral integration and barcoding of transplanted HSCs suggest that very low numbers of HSCs perpetuate a continuous stream of differentiating cells. However, the numbers of productive HSCs during normal haematopoiesis, and the flux of differentiating progeny remain unknown. Here we devise a mouse model allowing inducible genetic labelling of the most primitive Tie2(+) HSCs in bone marrow, and quantify label progression along haematopoietic development by limiting dilution analysis and data-driven modelling. During maintenance of the haematopoietic system, at least 30% or ∼5,000 HSCs are productive in the adult mouse after label induction. However, the time to approach equilibrium between labelled HSCs and their progeny is surprisingly long, a time scale that would exceed the mouse's life. Indeed, we find that adult haematopoiesis is largely sustained by previously designated 'short-term' stem cells downstream of HSCs that nearly fully self-renew, and receive rare but polyclonal HSC input. By contrast, in fetal and early postnatal life, HSCs are rapidly used to establish the immune and blood system. In the adult mouse, 5-fluoruracil-induced leukopenia enhances the output of HSCs and of downstream compartments, thus accelerating haematopoietic flux. Label tracing also identifies a strong lineage bias in adult mice, with several-hundred-fold larger myeloid than lymphoid output, which is only marginally accentuated with age. Finally, we show that transplantation imposes severe constraints on HSC engraftment, consistent with the previously observed oligoclonal HSC activity under these conditions. Thus, we uncover fundamental differences between the normal maintenance of the haematopoietic system, its regulation by challenge, and its re-establishment after transplantation. HSC fate mapping and its linked modelling provide a quantitative framework for studying in situ the regulation of haematopoiesis in health and disease.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
In response to infections and stress, hematopoiesis rapidly enhances blood and immune cell production. The stage within the hematopoietic hierarchy that accounts for this regeneration is unclear ...under natural conditions in vivo. We analyzed by differentiation tracing, using inducible Tie2- or Flt3-driven Cre recombinase, the roles of mouse hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). During polymicrobial sepsis, HSCs responded transcriptionally and increased their proliferation and cell death, yet HSC differentiation rates remained at steady-state levels. HSC differentiation was also independent from the ablation of various cellular compartments—bleeding, the antibody-mediated ablation of granulocytes or B lymphocytes, and genetic lymphocyte deficiency. By marked contrast, the fate mapping of MPPs in polymicrobial sepsis identified these cells as a major source for accelerated myeloid cell production. The regulation of blood and immune cell homeostasis by progenitors rather than stem cells may ensure a rapid response while preserving the integrity of the HSC population.
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•We traced the differentiation of HSCs and MPPs in vivo during perturbed hematopoiesis•Polymicrobial sepsis was sensed by HSCs but did not accelerate HSC differentiation•Loss of myeloid, erythroid, or lymphoid cells did not accelerate HSC differentiation•Sepsis caused accelerated myelopoiesis from MPPs
The cellular source of hematopoietic recovery after a challenge has been a long-standing question. Fanti and colleagues trace the differentiation of HSCs and MPPs in vivo and find that neither sepsis nor targeted cell ablation causes increased HSC output. Rather, the accelerated differentiation and proliferation of MPPs drive enhanced myelopoiesis upon sepsis.
The paradigmatic hematopoietic tree model is increasingly recognized to be limited, as it is based on heterogeneous populations largely defined by non-homeostatic assays testing cell fate potentials. ...Here, we combine persistent labeling with time-series single-cell RNA sequencing to build a real-time, quantitative model of in vivo tissue dynamics for murine bone marrow hematopoiesis. We couple cascading single-cell expression patterns with dynamic changes in differentiation and growth speeds. The resulting explicit linkage between molecular states and cellular behavior reveals widely varying self-renewal and differentiation properties across distinct lineages. Transplanted stem cells show strong acceleration of differentiation at specific stages of erythroid and neutrophil production, illustrating how the model can quantify the impact of perturbations. Our reconstruction of dynamic behavior from snapshot measurements is akin to how a kinetoscope allows sequential images to merge into a movie. We posit that this approach is generally applicable to understanding tissue-scale dynamics at high resolution.
Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved > 200‐fold expansion of functional ...HSCs, but their molecular characterization has not been possible since the majority of cells are non‐HSCs and single cell‐initiated cultures have substantial clone‐to‐clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non‐HSCs in expansion cultures. By directly linking single‐clone functional transplantation data with single‐clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This “repopulation signature” (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.
Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of ...functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells in these cultures being non-HSCs and the fact that single cell-initiated cultures display substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with EPCR, we have identified an in vitro reporter strategy capable of exclusively identifying HSC-containing clones and prospectively separating HSCs from non-HSCs. Linking single clone functional transplantation data with single clone gene expression profiling, we also reveal the molecular profile of expanded HSCs which is more similar to actively cycling fetal liver HSCs than quiescent adult bone marrow HSCs from which they were derived. A gene expression signature derived from the molecular profiling identifies core molecular components of HSCs, including Prdm16, Fstl1 and Palld, that can further predict the functional outcome in transplantation assays. Taken together, this system represents a new tool to be used for a wide-range of functional screening and molecular experiments previously not possible to carry out due to limited HSC numbers.
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