Single cell RNA-seq identifies the origins of heterogeneity in efficient cell transdifferentiation and reprogramming

Forced transcription factor expression can transdifferentiate somatic cells into other specialised cell types or reprogram them into induced pluripotent stem cells (iPSCs) with variable efficiency. To better understand the heterogeneity of these processes, we used single-cell RNA sequencing to follow the transdifferentation of murine pre-B cells into macrophages as well as their reprogramming into iPSCs. Even in these highly efficient systems, there was substantial variation in the speed and path of fate conversion. We predicted and validated that these differences are inversely coupled and arise in the starting cell population, with Mychigh large pre-BII cells transdifferentiating slowly but reprogramming efficiently and Myclow small pre-BII cells transdifferentiating rapidly but failing to reprogram. Strikingly, differences in Myc activity predict the efficiency of reprogramming across a wide range of somatic cell types. These results illustrate how single cell expression and computational analyses can identify the origins of heterogeneity in cell fate conversion processes.

[1]  Chunhui Hou,et al.  RNA Helicase DDX5 Inhibits Reprogramming to Pluripotency by miRNA-Based Repression of RYBP and its PRC1-Dependent and -Independent Functions. , 2017, Cell stem cell.

[2]  Thomas Seidl,et al.  Changes in gene expression profiles in developing B cells of murine bone marrow. , 2002, Genome research.

[3]  M. Ashburner,et al.  Gene Ontology: tool for the unification of biology , 2000, Nature Genetics.

[4]  Katie J. Clowers,et al.  Nudt21 Controls Cell Fate by Connecting Alternative Polyadenylation to Chromatin Signaling , 2018, Cell.

[5]  T. Graf,et al.  Historical origins of transdifferentiation and reprogramming. , 2011, Cell stem cell.

[6]  Jieying Zhu,et al.  H3K9 methylation is a barrier during somatic cell reprogramming into iPSCs , 2012, Nature Genetics.

[7]  Thomas Vierbuchen,et al.  Direct conversion of fibroblasts to functional neurons by defined factors , 2010, Nature.

[8]  Eliah G. Overbey,et al.  Aligning single-cell developmental and reprogramming trajectories identifies molecular determinants of reprogramming outcome , 2017, bioRxiv.

[9]  Laleh Haghverdi,et al.  Batch effects in single-cell RNA-sequencing data are corrected by matching mutual nearest neighbors , 2018, Nature Biotechnology.

[10]  A. Consiglio,et al.  Efficient and rapid generation of induced pluripotent stem cells from human keratinocytes , 2008, Nature Biotechnology.

[11]  Lothar Reichel,et al.  Augmented Implicitly Restarted Lanczos Bidiagonalization Methods , 2005, SIAM J. Sci. Comput..

[12]  Howard Y. Chang,et al.  Hierarchical Mechanisms for Direct Reprogramming of Fibroblasts to Neurons , 2013, Cell.

[13]  P. Rigollet,et al.  Reconstruction of developmental landscapes by optimal-transport analysis of single-cell gene expression sheds light on cellular reprogramming , 2017, bioRxiv.

[14]  Fabian J. Theis,et al.  Diffusion maps for high-dimensional single-cell analysis of differentiation data , 2015, Bioinform..

[15]  J. C. Belmonte,et al.  Dedifferentiation, transdifferentiation and reprogramming: three routes to regeneration , 2011, Nature Reviews Molecular Cell Biology.

[16]  Michael A. Halbisen,et al.  OSKM Induce Extraembryonic Endoderm Stem Cells in Parallel to Induced Pluripotent Stem Cells , 2016, Stem cell reports.

[17]  T. Graf,et al.  Stepwise Reprogramming of B Cells into Macrophages , 2004, Cell.

[18]  Howard Y. Chang,et al.  The histone chaperone CAF-1 safeguards somatic cell identity , 2015, Nature.

[19]  K. Hochedlinger,et al.  Epigenetic reprogramming and induced pluripotency , 2009, Development.

[20]  I. Amit,et al.  Massively Parallel Single-Cell RNA-Seq for Marker-Free Decomposition of Tissues into Cell Types , 2014, Science.

[21]  Scott W. Lowe,et al.  Stem cells: The promises and perils of p53 , 2009, Nature.

[22]  Chi V Dang,et al.  MYC on the Path to Cancer , 2012, Cell.

[23]  Denis Thieffry,et al.  C/EBPα poises B cells for rapid reprogramming into induced pluripotent stem cells , 2013, Nature.

[24]  Rudolf Jaenisch,et al.  Single-gene transgenic mouse strains for reprogramming adult somatic cells , 2010, Nature Methods.

[25]  A. Heguy,et al.  Nascent Induced Pluripotent Stem Cells Efficiently Generate Entirely iPSC-Derived Mice while Expressing Differentiation-Associated Genes. , 2018, Cell reports.

[26]  Esteban Ballestar,et al.  A robust and highly efficient immune cell reprogramming system. , 2009, Cell stem cell.

[27]  Biswajyoti Sahu,et al.  The interaction landscape between transcription factors and the nucleosome , 2018, Nature.

[28]  G. Bhagat,et al.  Early-stage epigenetic modification during somatic cell reprogramming by Parp1 and Tet2 , 2012, Nature.

[29]  Philip R. Gafken,et al.  Myc influences global chromatin structure , 2006, The EMBO journal.

[30]  Rudolf Jaenisch,et al.  Mechanisms and models of somatic cell reprogramming , 2013, Nature Reviews Genetics.

[31]  Jason D. Buenrostro,et al.  Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules. , 2019, Cell stem cell.

[32]  Fabian J Theis,et al.  Diffusion pseudotime robustly reconstructs lineage branching , 2016, Nature Methods.

[33]  M. Araúzo-Bravo,et al.  Pluripotent stem cells induced from adult neural stem cells by reprogramming with two factors , 2008, Nature.

[34]  N. Neff,et al.  Dissecting direct reprogramming from fibroblast to neuron using single-cell RNA-seq , 2016, Nature.

[35]  I. Amit,et al.  Single-cell transcriptome conservation in cryopreserved cells and tissues , 2016, Genome Biology.

[36]  M. Mann,et al.  C/EBPα creates elite cells for iPSC reprogramming by upregulating Klf4 and increasing the levels of Lsd1 and Brd4 , 2016, Nature Cell Biology.

[37]  Takashi Aoi,et al.  Generation of Pluripotent Stem Cells from Adult Mouse Liver and Stomach Cells , 2008, Science.

[38]  A. Melnick,et al.  Pre-B cell receptor-mediated activation of BCL6 induces pre-B cell quiescence through transcriptional repression of MYC. , 2011, Blood.

[39]  J. Mesirov,et al.  The Molecular Signatures Database Hallmark Gene Set Collection , 2015 .

[40]  Denis Thieffry,et al.  C/EBPα Activates Pre-existing and De Novo Macrophage Enhancers during Induced Pre-B Cell Transdifferentiation and Myelopoiesis , 2015, Stem cell reports.

[41]  Cole Trapnell,et al.  Aligning Single-Cell Developmental and Reprogramming Trajectories Identifies Molecular Determinants of Myogenic Reprogramming Outcome. , 2018, Cell systems.

[42]  K. Brennand,et al.  Reprogramming of Pancreatic β Cells into Induced Pluripotent Stem Cells , 2008, Current Biology.

[43]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[44]  W. Huber,et al.  Myc Depletion Induces a Pluripotent Dormant State Mimicking Diapause , 2016, Cell.

[45]  Jonathan M. Mudge,et al.  Creating reference gene annotation for the mouse C57BL6/J genome assembly , 2015, Mammalian Genome.

[46]  John C. Marioni,et al.  Correcting batch effects in single-cell RNA sequencing data by matching mutual nearest neighbours , 2017, bioRxiv.

[47]  S. Heath,et al.  Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate. , 2018, Cell stem cell.

[48]  T. Enver,et al.  Forcing cells to change lineages , 2009, Nature.

[49]  B. Blencowe,et al.  Myc and SAGA rewire an alternative splicing network during early somatic cell reprogramming , 2015, Genes & development.

[50]  A. Hutchins,et al.  Models of global gene expression define major domains of cell type and tissue identity , 2017, Nucleic acids research.

[51]  Erez Lieberman Aiden,et al.  Myc Regulates Chromatin Decompaction and Nuclear Architecture during B Cell Activation. , 2017, Molecular cell.

[52]  J. Ernst,et al.  Cooperative Binding of Transcription Factors Orchestrates Reprogramming , 2017, Cell.

[53]  K. Dorshkind,et al.  A stromal cell line from myeloid long-term bone marrow cultures can support myelopoiesis and B lymphopoiesis. , 1987, Journal of immunology.

[54]  K. Hochedlinger,et al.  Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells , 2009, Nature Genetics.

[55]  H. Weintraub,et al.  Expression of a single transfected cDNA converts fibroblasts to myoblasts , 1987, Cell.

[56]  H. Schöler,et al.  Oct4 distribution and level in mouse clones: consequences for pluripotency. , 2002, Genes & development.

[57]  Y. Benjamini,et al.  Controlling the false discovery rate: a practical and powerful approach to multiple testing , 1995 .

[58]  Javier Quilez,et al.  Transcription factors orchestrate dynamic interplay between genome topology and gene regulation during cell reprogramming , 2017, Nature Genetics.

[59]  Charles Y. Lin,et al.  Transcriptional Amplification in Tumor Cells with Elevated c-Myc , 2012, Cell.

[60]  S. Heath,et al.  Transcription Factors Drive Tet2-Mediated Enhancer Demethylation to Reprogram Cell Fate , 2018, Cell stem cell.

[61]  Christophe Benoist,et al.  Transcriptomes of the B and T Lineages Compared by Multiplatform Microarray Profiling , 2011, The Journal of Immunology.

[62]  G. Pan,et al.  Resolution of Reprogramming Transition States by Single Cell RNA-Sequencing , 2017, bioRxiv.

[63]  Alessandro Vullo,et al.  Ensembl 2015 , 2014, Nucleic Acids Res..

[64]  Helga Thorvaldsdóttir,et al.  Molecular signatures database (MSigDB) 3.0 , 2011, Bioinform..

[65]  Roderic Guigó,et al.  The GEM mapper: fast, accurate and versatile alignment by filtration , 2012, Nature Methods.