Cell type of origin influences the molecular and functional properties of mouse induced pluripotent stem cells

Journal name:
Nature Biotechnology
Volume:
28,
Pages:
848–855
Year published:
DOI:
doi:10.1038/nbt.1667
Received
Accepted
Published online

Abstract

Induced pluripotent stem cells (iPSCs) have been derived from various somatic cell populations through ectopic expression of defined factors. It remains unclear whether iPSCs generated from different cell types are molecularly and functionally similar. Here we show that iPSCs obtained from mouse fibroblasts, hematopoietic and myogenic cells exhibit distinct transcriptional and epigenetic patterns. Moreover, we demonstrate that cellular origin influences the in vitro differentiation potentials of iPSCs into embryoid bodies and different hematopoietic cell types. Notably, continuous passaging of iPSCs largely attenuates these differences. Our results suggest that early-passage iPSCs retain a transient epigenetic memory of their somatic cells of origin, which manifests as differential gene expression and altered differentiation capacity. These observations may influence ongoing attempts to use iPSCs for disease modeling and could also be exploited in potential therapeutic applications to enhance differentiation into desired cell lineages.

At a glance

Figures

  1. iPSCs derived from different cell types are transcriptionally distinguishable.
    Figure 1: iPSCs derived from different cell types are transcriptionally distinguishable.

    (a) Flow chart explaining the derivation and analysis of genetically matched iPSCs from different cell types. Secondary iPSCs were first injected into blastocysts to generate chimeric mice, from which the indicated somatic cell types were isolated. Exposure of these cells to doxycycline (dox) then gave rise to iPSCs. ChIP, chromatin immunoprecipitation. (b) Quantification of the expression levels of Cxcr4, Itgb1, Gr-1 and Lysozyme by quantitative PCR in SMP-iPSCs, in red, and Gra-iPSCs, in gray. The values were normalized to GAPDH expression; the error bars depict the s.e.m. (n = 3). (c) Heat map showing top 104 probes with highest variance in their expression levels. Left panel, SMP-iPSCs and Gra-iPSCs derived from chimera no. 1. Right panel, TTF-iPSCs and B-iPSCs derived from chimera no. 2. (d) Hierarchical, unsupervised clustering of iPSC expression profiles using the correlation distance and the Ward method. SMP-iPSCs and Gra-iPSCs were derived from chimera no. 1 (left panel), TTF-iPSCs and B-iPSCs originate from chimera no. 2 (right panel). Chi no. 1, chimera no. 1; chi no. 2, chimera no. 2.

  2. iPSCs derived from different cell types exhibit distinguishable epigenetic signatures.
    Figure 2: iPSCs derived from different cell types exhibit distinguishable epigenetic signatures.

    (a) Hierarchical unsupervised clustering analysis of HELP genome-wide methylation data from indicated iPSC lines. (b) Correspondence analysis of SMP-iPSCs and Gra-iPSCs (left panel) from chimera no. 1, TTF-iPSCs and B-iPSCs (right panel) from chimera no. 2. (c) Graphic representation of DNA methylation quantification of specific CpGs (circles) in the promoter regions of the indicated candidate genes using EpiTYPER DNA methylation analyses. Yellow indicates 0% methylation and blue 100% methylation. (d) Chromatin immunoprecipitation (ChIP) for H3 pan-acetylated (H3Ac, in blue), H3K4 trimethylated (H3K4me3, in green), H3K27 trimethylated (H3K27me3, in red) and isotype control (IgG, in light blue) of granulocytes (Gra), SMPs, Gra-iPSCs and SMP-iPSCs. Chi no. 1, chimera no. 1; chi no. 2, chimera no. 2. The error bars depict the s.e.m. (n = 3).

  3. iPSCs derived from different cell types have distinctive in vitro differentiation potentials.
    Figure 3: iPSCs derived from different cell types have distinctive in vitro differentiation potentials.

    (a) Experimental outline. iPSCs were first differentiated into embryoid bodies. At day 6, embryoid bodies were dissociated and plated in conditions to favor differentiation into erythrocyte progenitors (eryP) and macrophage and mixed hematopoietic colonies. (b) Phase contrast images showing embryoid bodies derived from B-iPSCs, TTF-iPSCs, Gra-iPSCs and SMP-iPSCs at same magnification. (c) Quantification of embryoid body sizes derived from B-iPSCs, TTF-iPSCs, Gra-iPSCs and SMP-iPSCs; the diameter of the embryoid bodies was measured using arbitrary units (AU). The error bars depict the s.e.m. (n = 30) (d) Representative images of erythrocyte progenitors (eryPs), macrophage colonies and mixed hematopoietic colonies. (eg) Quantification of in vitro differentiation potentials of the different iPSCs into EryPs (e), macrophage colonies (f) and mixed hematopoietic colonies (g). Chi no. 1, chimera no. 1; chi no. 2, chimera no. 2. The error bars depict the s.e.m. (n = 12).

  4. Continuous passaging of iPSCs abrogates transcriptional, epigenetic and functional differences.
    Figure 4: Continuous passaging of iPSCs abrogates transcriptional, epigenetic and functional differences.

    (a) Hierarchical unsupervised clustering of expression profiles from B-iPSCs, T-iPSCs, TTF-iPSCs and Gra-iPSCs from chimera no. 2. Left panel shows clustering analysis of all iPSC samples at passage p4, the middle panel at p10 and the right panel at p16. (b) Number of differentially expressed probes between pairs of iPSC samples used in a; iPSCs at p4 are shown in blue bars, iPSCs at p10 are shown in orange bars and iPSCs at p16 are shown in red bars. The number of differently expressed probes between iPSCs was calculated using a pairwise analysis (twofold), with t-test P = 0.05, with Bejamini and Hochberg correction (n = 3). (c) Venn diagram and GO analysis showing overlap of genes that change from p4 to p16 in Gra-IPSCs, TTF-iPSCs and B-iPSCs. Red line marks functional GO cluster of genes shared between all three iPSC groups. Black line marks functional GO cluster of genes shared by at least two of the iPSC groups. Functional ontology cluster analysis was performed using the DAVIS algorithm. (d) Hierarchical unsupervised clustering using HELP genome-wide methylation profiles of B-iPSCs and TTF-iPSCs at p16. (eg) Quantification of in vitro differentiation potentials of B-iPSCs and TTF-iPSCs at p16 into EryPs (e), macrophage colonies (f) and mixed hematopoietic colonies (g). The error bars depict the s.e.m. (n = 9).

  5. Model summarizing the presented data.
    Figure 5: Model summarizing the presented data.

    iPSCs derived from different somatic cell types retain a transient epigenetic and transcriptional memory of their cell type of origin at early passage, despite acquiring pluripotent gene expression, transgene-independent growth and the ability to contribute to tissues in chimeras. Continuous passaging resolves these differences, giving rise to iPSCs that are molecularly and functionally indistinguishable. Note the difference between early passage iPSCs and partially reprogrammed cells, which require continuous viral transgene expression and fail to activate endogenous pluripotency genes or support the development of viable mice.

Accession codes

Primary accessions

Gene Expression Omnibus

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Author information

Affiliations

  1. Howard Hughes Medical Institute and Department of Stem Cell and Regenerative Biology, Harvard University and Harvard Medical School, Cambridge, Massachusetts, USA.

    • Jose M Polo,
    • Warakorn Kulalert,
    • Sarah Eminli,
    • Kah Yong Tan,
    • Effie Apostolou,
    • Matthias Stadtfeld,
    • Amy J Wagers &
    • Konrad Hochedlinger
  2. Massachusetts General Hospital Cancer Center, Charlestown, Massachusetts, USA.

    • Jose M Polo,
    • Warakorn Kulalert,
    • Sarah Eminli,
    • Effie Apostolou,
    • Matthias Stadtfeld,
    • Toshi Shioda &
    • Konrad Hochedlinger
  3. Massachusetts General Hospital Center for Regenerative Medicine, Boston, Massachusetts, USA.

    • Jose M Polo,
    • Warakorn Kulalert,
    • Sarah Eminli,
    • Effie Apostolou,
    • Matthias Stadtfeld &
    • Konrad Hochedlinger
  4. Harvard Stem Cell Institute, Cambridge, Massachusetts, USA.

    • Jose M Polo,
    • Warakorn Kulalert,
    • Sarah Eminli,
    • Kah Yong Tan,
    • Effie Apostolou,
    • Matthias Stadtfeld,
    • Amy J Wagers &
    • Konrad Hochedlinger
  5. Department of Surgery, Weill Cornell Medical College, New York, New York, USA.

    • Susanna Liu &
    • Todd Evans
  6. Department of Medicine, Hematology Oncology Division, Weill Cornell Medical College, New York, New York, USA.

    • Maria Eugenia Figueroa,
    • Yushan Li &
    • Ari Melnick
  7. Sanofi-Aventis Cambridge Genomics Center, Cambridge, Massachusetts, USA.

    • Sridaran Natesan
  8. Joslin Diabetes Center, Boston, Massachusetts, USA.

    • Kah Yong Tan &
    • Amy J Wagers

Contributions

J.M.P. and K.H. conceived the study, interpreted results and wrote the manuscript; J.M.P. performed most of the experiments with help from W.K.; S.L. and T.E. performed and interpreted in vitro differentiation assays; M.E.F and A.M. performed and analyzed HELP methylation experiments; K.Y.T. and A.J.W. isolated SMPs and derived most SMP-iPSCs; T.S. and S.N. performed expression arrays; and S.E., E.A. and M.S. provided essential study material. All authors gave critical input to the manuscript draft.

Competing financial interests

K.H. is an advisor for iPierian.

Corresponding author

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Supplementary information

PDF files

  1. Supplementary Text and Figures (6M)

    Supplementary Figures 1–13 and Supplementary Table 1

Excel files

  1. Supplementary Table 2 (108K)

    Accession numbers of differentially expressed genes between indicated pairs of iPSCs.

  2. Supplementary Table 3 (16K)

    Probe-set names and gene symbols of differentially methylated genes between SMP-iPSC and Gra-iPSC.

  3. Supplementary Table 4 (20K)

    List of primers used for Q-PCR and Q-ChIP analyses.

  4. Supplementary Table 5 (12K)

    List of primers used for Mass Array Epityping.

Additional data