Single-cell RNA sequencing studies of differentiating systems have raised fundamental questions regarding the discrete versus continuous nature of both differentiation and cell fate. Here we present Palantir, an algorithm that models trajectories of differentiating cells by treating cell fate as a probabilistic process and leverages entropy to measure cell plasticity along the trajectory. Palantir generates a high-resolution pseudo-time ordering of cells and, for each cell state, assigns a probability of differentiating into each terminal state. We apply our algorithm to human bone marrow single-cell RNA sequencing data and detect important landmarks of hematopoietic differentiation. Palantir’s resolution enables the identification of key transcription factors that drive lineage fate choice and closely track when cells lose plasticity. We show that Palantir outperforms existing algorithms in identifying cell lineages and recapitulating gene expression trends during differentiation, is generalizable to diverse tissue types, and is well-suited to resolving less-studied differentiating systems.
Subscribe to Journal
Get full journal access for 1 year
only $4.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Palantir is available as a Python module at https://github.com/dpeerlab/Palantir/. A Jupyter notebook detailing the workflow including data preprocessing, running Palantir along with a demonstration of various plots, and visualizations is available at http://nbviewer.jupyter.org/github/dpeerlab/Palantir/blob/master/notebooks/Palantir_sample_notebook.ipynb. The code and data for this article, along with an accompanying computational environment, are available and executable online as a Code Ocean capsule: https://doi.org/10.24433/CO.6f3a9d2b-82d6-45bd-a583-5346a30e0c5d (ref. 58).
Raw and processed data are available through the Human Cell Atlas data portal at https://data.humancellatlas.org/explore/projects/091cf39b-01bc-42e5-9437-f419a66c8a45.
Bendall, S. C. et al. Single-cell trajectory detection uncovers progression and regulatory coordination in human B cell development. Cell 157, 714–725 (2014).
Setty, M. et al. Wishbone identifies bifurcating developmental trajectories from single-cell data. Nat. Biotechnol. 34, 637–645 (2016).
Haghverdi, L., Buttner, M., Wolf, F. A., Buettner, F. & Theis, F. J. Diffusion pseudotime robustly reconstructs lineage branching. Nat. Methods 13, 845–848 (2016).
Velten, L. et al. Human haematopoietic stem cell lineage commitment is a continuous process. Nat. Cell Biol. 19, 271–281 (2017).
Buenrostro, J. D. et al. Integrated single-cell analysis maps the continuous regulatory landscape of human hematopoietic differentiation. Cell 173, 1535–1548 e1516 (2018).
Paul, F. et al. Transcriptional heterogeneity and lineage commitment in myeloid progenitors. Cell 163, 1663–1677 (2015).
Plass, M. et al. Cell type atlas and lineage tree of a whole complex animal by single-cell transcriptomics. Science 360, eaaq1723 (2018).
Street, K. et al. Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics. BMC Genomics 19, 477 (2018).
Stergachis, A. B. et al. Developmental fate and cellular maturity encoded in human regulatory DNA landscapes. Cell 154, 888–903 (2013).
Corces, M. R. et al. Lineage-specific and single-cell chromatin accessibility charts human hematopoiesis and leukemia evolution. Nat. Genet. 48, 1193–1203 (2016).
Orkin, S. H. & Zon, L. I. Hematopoiesis: an evolving paradigm for stem cell biology. Cell 132, 631–644 (2008).
Amir el, A. D. et al. viSNE enables visualization of high dimensional single-cell data and reveals phenotypic heterogeneity of leukemia. Nat. Biotechnol. 31, 545–552 (2013).
Levine, J. H. et al. Data-driven phenotypic dissection of AML reveals progenitor-like cells that correlate with prognosis. Cell 162, 184–197 (2015).
Coifman, R. R. et al. Geometric diffusions as a tool for harmonic analysis and structure definition of data: diffusion maps. Proc. Natl Acad. Sci. USA 102, 7426–7431 (2005).
Haber, A. L. et al. A single-cell survey of the small intestinal epithelium. Nature 551, 333–339 (2017).
Hastie, T. J. & Tibshirani, R. J. Generalized Additive Models (Chapman & Hall/CRC, 1990).
Qiu, X. et al. Reversed graph embedding resolves complex single-cell trajectories. Nat. Methods 14, 979–982 (2017).
Dahlin, J. S. et al. A single-cell hematopoietic landscape resolves 8 lineage trajectories and defects in Kit mutant mice. Blood 131, e1–e11 (2018).
Azizi, E. et al. Single-cell map of diverse immune phenotypes in the breast tumor microenvironment. Cell 174, 1293–1308.e36 (2018).
Novershtern, N. et al. Densely interconnected transcriptional circuits control cell states in human hematopoiesis. Cell 144, 296–309 (2011).
Psaila, B. et al. Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways. Genome Biol. 17, 83 (2016).
Pei, W. et al. Polylox barcoding reveals haematopoietic stem cell fates realized in vivo. Nature 548, 456–460 (2017).
Takubo, K. et al. Regulation of glycolysis by Pdk functions as a metabolic checkpoint for cell cycle quiescence in hematopoietic stem cells. Cell Stem Cell 12, 49–61 (2013).
Majeti, R., Park, C. Y. & Weissman, I. L. Identification of a hierarchy of multipotent hematopoietic progenitors in human cord blood. Cell Stem Cell 1, 635–645 (2007).
Mori, Y. et al. Identification of the human eosinophil lineage-committed progenitor: revision of phenotypic definition of the human common myeloid progenitor. J. Exp. Med. 206, 183–193 (2009).
Ravet, E. et al. Characterization of DNA-binding-dependent and -independent functions of SCL/TAL1 during human erythropoiesis. Blood 103, 3326–3335 (2004).
Siatecka, M. & Bieker, J. J. The multifunctional role of EKLF/KLF1 during erythropoiesis. Blood 118, 2044–2054 (2011).
Ferreira, R., Ohneda, K., Yamamoto, M. & Philipsen, S. GATA1 function, a paradigm for transcription factors in hematopoiesis. Mol. Cell. Biol. 25, 1215–1227 (2005).
Funnell, A. P. et al. Erythroid Kruppel-like factor directly activates the basic Kruppel-like factor gene in erythroid cells. Mol. Cell. Biol. 27, 2777–2790 (2007).
Nerlov, C., Querfurth, E., Kulessa, H. & Graf, T. GATA-1 interacts with the myeloid PU.1 transcription factor and represses PU.1-dependent transcription. Blood 95, 2543–2551 (2000).
Zhang, P. et al. PU.1 inhibits GATA-1 function and erythroid differentiation by blocking GATA-1 DNA binding. Blood 96, 2641–2648 (2000).
May, G. et al. Dynamic analysis of gene expression and genome-wide transcription factor binding during lineage specification of multipotent progenitors. Cell Stem Cell 13, 754–768 (2013).
Tusi, B. K. et al. Population snapshots predict early haematopoietic and erythroid hierarchies. Nature 555, 54–60 (2018).
Antebi, Y. E. et al. Mapping differentiation under mixed culture conditions reveals a tunable continuum of T cell fates. PLoS Biol. 11, e1001616 (2013).
Herring, C. A. et al. Unsupervised trajectory analysis of single-cell RNA-seq and imaging data reveals alternative tuft cell origins in the gut. Cell Syst. 6, 37–51 e39 (2018).
Li, H. & Jasper, H. Gastrointestinal stem cells in health and disease: from flies to humans. Dis. Model Mech. 9, 487–499 (2016).
Herman, J. S., Sagar & Grun, D. FateID infers cell fate bias in multipotent progenitors from single-cell RNA-seq data. Nat. Methods 15, 379–386 (2018).
Yang, J., Zhang, L., Yu, C., Yang, X. F. & Wang, H. Monocyte and macrophage differentiation: circulation inflammatory monocyte as biomarker for inflammatory diseases. Biomark. Res. 2, 1 (2014).
Benschop, R. J. & Cambier, J. C. B cell development: signal transduction by antigen receptors and their surrogates. Curr. Opin. Immunol. 11, 143–151 (1999).
Merad, M., Sathe, P., Helft, J., Miller, J. & Mortha, A. The dendritic cell lineage: ontogeny and function of dendritic cells and their subsets in the steady state and the inflamed setting. Annu. Rev. Immunol. 31, 563–604 (2013).
Hoppe, P. S. et al. Early myeloid lineage choice is not initiated by random PU.1 to GATA1 protein ratios. Nature 535, 299–302 (2016).
Ibarra-Soria, X. et al. Defining murine organogenesis at single-cell resolution reveals a role for the leukotriene pathway in regulating blood progenitor formation. Nat. Cell Biol. 20, 127–134 (2018).
Regev, A. et al. The human cell atlas. Elife 6, e27041 (2017).
Farrell, J. A. et al. Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis. Science 360, 981–987 (2018).
Kotton, D. N. & Morrisey, E. E. Lung regeneration: mechanisms, applications and emerging stem cell populations. Nat. Med. 20, 822–832 (2014).
Beck, B. & Blanpain, C. Unravelling cancer stem cell potential. Nat. Rev. Cancer 13, 727–738 (2013).
Raj, B. et al. Simultaneous single-cell profiling of lineages and cell types in the vertebrate brain. Nat. Biotechnol. 36, 442–450 (2018).
Spanjaard, B. et al. Simultaneous lineage tracing and cell-type identification using CRISPR–Cas9-induced genetic scars. Nat. Biotechnol. 36, 469–473 (2018).
Biezuner, T. et al. A generic, cost-effective, and scalable cell lineage analysis platform. Genome Res. 26, 1588–1599 (2016).
Macaulay, I. C. et al. G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat. Methods 12, 519–522 (2015).
van Dijk, D. et al. Recovering gene interactions from single-cell data using data diffusion. Cell 174, 716–729.e27 (2018).
Finak, G. et al. MAST: a flexible statistical framework for assessing transcriptional changes and characterizing heterogeneity in single-cell RNA sequencing data. Genome Biol. 16, 278 (2015).
Klein, A. M. et al. Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells. Cell 161, 1187–1201 (2015).
Buettner, F. et al. Computational analysis of cell-to-cell heterogeneity in single-cell RNA-sequencing data reveals hidden subpopulations of cells. Nat. Biotechnol. 33, 155–160 (2015).
Buettner, F., Pratanwanich, N., McCarthy, D. J., Marioni, J. C. & Stegle, O. f-scLVM: scalable and versatile factor analysis for single-cell RNA-seq. Genome Biol. 18, 212 (2017).
van der Maaten, L. P. J. & Hinton, G. E. Visualizing high-dimensional data using t-SNE. J. Mach. Learn. Res. 9, 2579–2605 (2008).
Wolf, F. A., Angerer, P. & Theis, F. J. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol. 19, 15 (2018).
Setty, M. et al. Characterization of cell fate probabilities in single-cell data with Palantir. Code Ocean Capsule https://doi.org/10.24433/CO.6f3a9d2b-82d6-45bd-a583-5346a30e0c5d (2018).
We thank R. Sharma for valuable conversations related to this manuscript, C. Trasande and T. Nawy for helping to write the manuscript, and E. Azizi, C. Burdziak, and K. Hadjantonakis for valuable comments. This study was supported by NIH grants nos. NIH DP1-HD084071 and NIH R01CA164729, Cancer Center Support Grant no. P30 CA008748, and the Gerry Center for Metastasis and Tumor Ecosystems.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Setty, M., Kiseliovas, V., Levine, J. et al. Characterization of cell fate probabilities in single-cell data with Palantir. Nat Biotechnol 37, 451–460 (2019). https://doi.org/10.1038/s41587-019-0068-4
Single-cell transcriptome atlas and chromatin accessibility landscape reveal differentiation trajectories in the rice root
Nature Communications (2021)
Fully defined human pluripotent stem cell-derived microglia and tri-culture system model C3 production in Alzheimer’s disease
Nature Neuroscience (2021)
Nature Computational Science (2021)
Single-cell profiling of the human decidual immune microenvironment in patients with recurrent pregnancy loss
Cell Discovery (2021)
Evaluating genetic causes of azoospermia: What can we learn from a complex cellular structure and single-cell transcriptomics of the human testis?
Human Genetics (2021)