Direct pericyte-to-neuron reprogramming via unfolding of a neural stem cell-like program

Abstract

Ectopic expression of defined transcription factors can force direct cell-fate conversion from one lineage to another in the absence of cell division. Several transcription factor cocktails have enabled successful reprogramming of various somatic cell types into induced neurons (iNs) of distinct neurotransmitter phenotype. However, the nature of the intermediate states that drive the reprogramming trajectory toward distinct iN types is largely unknown. Here we show that successful direct reprogramming of adult human brain pericytes into functional iNs by Ascl1 and Sox2 encompasses transient activation of a neural stem cell-like gene expression program that precedes bifurcation into distinct neuronal lineages. During this transient state, key signaling components relevant for neural induction and neural stem cell maintenance are regulated by and functionally contribute to iN reprogramming and maturation. Thus, Ascl1- and Sox2-mediated reprogramming into a broad spectrum of iN types involves the unfolding of a developmental program via neural stem cell-like intermediates.

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Fig. 1: Ascl1–Sox2 synergism is required for pericyte-to-iN reprogramming.
Fig. 2: Pericyte heterogeneity correlates with distinct reprogramming competence.
Fig. 3: A transient neural precursor-like state emerges on the reprogramming path to iNs.
Fig. 4: Modulation of signaling pathways identified during neural stem cell-like state.
Fig. 5: Pericytes give rise to distinct neuronal subtypes, and targeting BMP signaling promotes maturation.

Change history

  • 15 August 2018

    In the version of this article initially published online, Supplementary Table 7 could not be opened. The error has been corrected online.

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Acknowledgements

We thank M. Wernig (Stanford University) for generously providing us with the Sox2 coding sequence. We are also very grateful to B. Sutor (BMC, LMU Munich) for help with the electrophysiological experiments, R. Menon (UMC Mainz) for help with the cell culture experiments, A. Bosio (Miltenyi Biotec) for help with the multidimensional fluorescence stainings, and F. Calzolari (UMC Mainz) for comments on the manuscript. We thank B. Höber, A. Weihmann, and J. Kelso of MPI-EVA for sequencing and bioinformatics support with this project. Flow cytometric cell sorting was performed at the “Core Unit Durchflusszytometrie” (CUDZ) of the Center for Infectious Diseases at the College of Veterinary Medicine, University of Leipzig, Leipzig, Germany. S.F. was supported by a fellowship from the Swiss National Science Foundation (PA00P3_139709). W.F. was supported by a Fellowship from the China Research Council. This work was supported by the following grants: advanced ERC ChroNeuroRepair to M.G.; Bavarian State Ministry of Sciences, Research and the Arts to M.K. and B.B. (ForIPS D2-F2412.26); Schram foundation (T287/29577/2017) and Wings For Life (WFL-DE-012/14) to M.K.; Max Planck Society to B.T.; and DFG (INST 161/875-2; BE 4182/8-1), NEURON ERA-NET (01EW1604), Wellcome Trust (206410/Z/17/Z), and the research initiative “Wissen schafft Zukunft” of Rhineland-Palatinate to B.B.

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Contributions

M.K., J.G.C., B.T., and B.B. conceived the study and designed experiments; M.K. performed direct reprogramming experiments; M.K., S.F., A.P., and V.K.T. analyzed bulk RNA-seq data; A.B. helped with processing of the 10xGenomics data; A.G. performed RNA isolation for bulk RNA-seq analysis; W.F. performed time-lapse imaging experiments; T.R. performed electrophysiological recordings; A.C. performed Sholl analyses; A.S. performed immunocytochemical analyses; C.S. provided human brain biopsies; M.G. provided material; J.G.C., M.G.-S., and T.G. performed single-cell RNA-seq experiments and sequenced libraries; J.G.C., J.K., and B.T. analyzed single-cell RNA-seq data; all authors discussed the data; and M.K., J.G.C., S.F., B.T., and B.B. wrote the paper.

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Correspondence to Marisa Karow or Barbara Treutlein or Benedikt Berninger.

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Integrated supplementary information

Supplementary Figure 1 Cooperative AS function is required for pericyte-to-iN conversion.

a, Euler diagram shows number of DE genes of each condition (Ascl1-only, Sox2-only, AS) at 2 dpi. b, Bar graph representing number of direct Ascl1-targets transcriptionally upregulated across different conditions and timepoints. c, Euler diagram showing that the majority of direct Ascl1-targets are regulated by AS synergism in human brain pericytes at 2 and 7 dpi. d, Heatmap shows normalized expression (Z-score) of direct Ascl1-target genes upregulated in AS-transduced cells at 7 dpi. Note that only a minor fraction of these genes is also upregulated by Ascl1 alone. e, GO term analysis of DE genes for each condition at 2 dpi. Shown are the 10 most significantly regulated GO terms. GO terms were ordered according to their significance as determined by Fisher´s exact test; n = 3 individual pericyte donors per experimental group. f, GO term analysis of DE genes for each condition at 7 dpi. Significantly regulated genes in GO categories are contained in Supplementary Table 2. GO terms were ordered according to their significance as determined by Fisher´s exact test; n = 3 individual pericyte donors per experimental group. g, tSNE plots from the analysis of Ascl1-only and AS transduced cells at 2 and 7 dpi from Fig. 1e are colored based on the expression of selected pericyte genes (Log2 FPKM). h, tSNE plots from the analysis of Ascl1-only and AS transduced cells at 2 and 7 dpi from Fig. 1e are colored based on the expression of selected mesoderm, neurogenesis-related, and GABAergic signature genes (Log2 FPKM).

Supplementary Figure 2 Expression of pericyte heterogeneity genes.

a, tSNE plot from Fig. 2a colored based on the expression of selected genes enriched in different pericyte groups. b, Violin plots show the density expression distribution of genes in pericyte group 1 (31 cells) and group 2 (44 cells). c, GO term analysis of pericyte heterogeneity genes from Fig. 2b; group 1, n = 31 cells; group 2, n = 44 cells; GO terms were ordered according to their significance as determined by Fisher´s exact test. d, Representative flow cytometry plot showing isotype control for the sorting of LEPR-positive cultured human pericytes (Fig. 2f); n = 4.

Supplementary Figure 3 Switch genes are expressed in the germinal zones of the developing CNS.

a In situ hybridization images (GenePaint.org) show the expression of selected switch genes in the developing mouse forebrain (E14.5). b Projecting the switch gene signature onto published scRNA-seq data1 reconstructing the MEF-to-iN reprogramming path (259 cells) shows the high base level in the starting MEF population and downregulation of the switch gene signature along differentiation towards iNs. Shaded gray represents 0.95 confidence interval.

Supplementary Figure 4 Characterization of ASD cells and shift towards a glutamatergic neuron phenotye by combinatorial expression of AS with Neurog2.

a, Summary of morphometric analyses showing significant differences between untreated and Dorsomorphin-treated AS cells. Data are shown as mean ± SEM; (untreated, n = 14 cells of 3 independent experiments; Dorsomorphin-treated, n = 14 cells of 3 independent experiments); two-tailed unpaired Student's t test; primary branches, P = 0.015; number of dendritic segments, P = 0.0005; branching points, P = 0.001; maximum length, P = 0.035; soma size, P = 0.0006; sum of intersections P = 0.02; *P < 0.05, ** P < 0.01, ***P < 0.001. b, Electrophysiological assessment of AS and ASD cells. Representative traces of multiple action potential discharge after step-current injections in AS (left) and ASD (middle) cells. Action potentials could be reliably blocked by TTX [0.5 µM] bath application (right). c, Micrographs show iGNs treated with Dorsomorphin immunoreactive for TUBB3 and GABA which acquire highly complex neuronal morphology. ASD iGNs show overlapping immunoreactivity for parvalbumin (PVALB) and GABA (n =3). Nuclei are stained with Dapi. Scale bars = 50 µm. d, Biplot showing the expression of DLX1 and NEUROG2 in all SNAP25-expressing cells. e, Monocle plot from Fig. 4g colored based on the expression of genes that show the loss of pericyte marker gene expression along the pseudotime and the acquisition of GABAergic and glutamatergic cell fate determinants along the trajectory towards different neuronal subtypes. f, Schematic of ASN experiments. g, Micrographs show pericytes transduced with AS (green) and Neurog2 (red) that acquire VGLUT1 immunoreactivity by co-expression of these three transcription factors. Note the punctate appearance of VGLUT1 in inset (right) (n = 3). Nuclei are stained with Dapi. Scale bars = 50 µm.

Supplementary Figure 5 High-throughput scRNA-seq data from a second pericyte donor confirms lineage bifurcations during iN maturation.

a, scRNA-seq using a high-throughput droplet microfluidic platform (10X genomics) was performed on 3419 AS-transduced cells treated with Dorsomorphin at 14 days post infection. PCA followed by tSNE shows cell populations that maintain pericyte markers (greys) and fail to productively differentiate to a neuronal lineage, populations at intermediate stages of differentiation (light grey), and two distinct neuron populations (cyan, blue). b, Cells are colored in the tSNE plots based on log normalized expression of pericyte marker PDGFRB, group 1 marker LEPR, and neuronal marker SNAP25. c, The inset shows the log normalized expression of markers for distinct excitatory (NEUROG2) and inhibitory (DLX1) neuronal populations that emerge during reprogramming. d, Heatmap shows the scaled expression of the top 20 genes that are differentially expressed (based on average log fold change) between the two neuronal populations. Single cells are in columns, genes in rows.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5

Reporting Summary

Supplementary Table 1

DE analysis of bulk RNA-sequencing data following overexpression of Ascl1, Sox2, Ascl1-Sox2, and control vector.

Supplementary Table 2

GO terms including significant genes within GO terms of DE genes from bulk RNA-seq analysis.

Supplementary Table 3

Comparison of DE genes with direct Ascl1-targets.

Supplementary Table 4

Genes in GO terms of pericyte group 1 and group 2.

Supplementary Table 5

Fluidigm C1 transcriptome data for all 769 cells with annotations (quantification in log2[FPKM]).

Supplementary Table 6

Genes used for calculating pericyte, mesodermal, neurogenesis-related, and GABAergic signatures.

Supplementary Table 7

10x Genomics transcriptome data for all 3419 sorted ASD cells.

Supplementary Video 1

Time-lapse video microscopy of human brain pericytes transduced with AS and treated with Dorsomorphin.

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Karow, M., Camp, J.G., Falk, S. et al. Direct pericyte-to-neuron reprogramming via unfolding of a neural stem cell-like program. Nat Neurosci 21, 932–940 (2018). https://doi.org/10.1038/s41593-018-0168-3

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