Dedifferentiation of committed epithelial cells into stem cells in vivo (original) (raw)

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Acknowledgements

The work in this manuscript was supported by a Harvard Stem Cell Institute Seed Grant and a National Institutes of Health-National Heart, Lung, and Blood Institute Early Career Research New Faculty (P30) award (5P30HL101287-02) and a Harvard Stem Cell Institute (HSCI) Junior Investigator Grant to J.R. J.R. is a New York Stem Cell Foundation-Robertson Investigator. We wish to extend our thanks to W. Anderson, Y. Dor, Q. Zhou, A. Brack, J. Galloway and all members of the Rajagopal laboratory for their constructive criticism. We thank the members of the HSCI flow cytometry core facility for help with cell sorting.

Author information

Authors and Affiliations

  1. Center for Regenerative Medicine, Massachusetts General Hospital, 185 Cambridge Street, Boston, Massachusetts 02114, USA,
    Purushothama Rao Tata, Hongmei Mou, Ana Pardo-Saganta, Rui Zhao, Mythili Prabhu, Brandon M. Law, Vladimir Vinarsky, Amar Sahay & Jayaraj Rajagopal
  2. Departments of Pediatrics, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA
    Purushothama Rao Tata, Hongmei Mou, Ana Pardo-Saganta, Rui Zhao, Mythili Prabhu, Brandon M. Law, Vladimir Vinarsky & Jayaraj Rajagopal
  3. Department of Internal Medicine, Pulmonary and Critical Care Unit, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA
    Purushothama Rao Tata, Hongmei Mou, Ana Pardo-Saganta, Rui Zhao, Mythili Prabhu, Brandon M. Law, Vladimir Vinarsky, Josalyn L. Cho, Benjamin D. Medoff & Jayaraj Rajagopal
  4. Harvard Stem Cell Institute, Cambridge, 02138, Massachusetts, USA
    Purushothama Rao Tata, Hongmei Mou, Ana Pardo-Saganta, Rui Zhao, Mythili Prabhu, Brandon M. Law, Vladimir Vinarsky, Amar Sahay & Jayaraj Rajagopal
  5. Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, 02129, Massachusetts, USA
    Josalyn L. Cho & Benjamin D. Medoff
  6. Program in Membrane Biology and Nephrology Division, Center for Systems Biology, Massachusetts General Hospital and Harvard Medical School, Boston, 02214, Massachusetts, USA
    Sylvie Breton
  7. Department of Psychiatry, Harvard Medical School, Boston, 02215, Massachusetts, USA
    Amar Sahay

Authors

  1. Purushothama Rao Tata
  2. Hongmei Mou
  3. Ana Pardo-Saganta
  4. Rui Zhao
  5. Mythili Prabhu
  6. Brandon M. Law
  7. Vladimir Vinarsky
  8. Josalyn L. Cho
  9. Sylvie Breton
  10. Amar Sahay
  11. Benjamin D. Medoff
  12. Jayaraj Rajagopal

Contributions

P.R.T. designed and performed experiments and wrote the manuscript; H.M., A.P.-S., R.Z., M.P., B.M.L. and V.V. performed ex vivo experiments; J.L.C. performed influenza infection experiments; A.S. provided tet(O)DTA mice and edited the manuscript; S.B. provided B1-eGFP mice; B.D.M. reviewed the manuscript; J.R. suggested and co-designed the study and co-wrote the manuscript with P.R.T.

Corresponding author

Correspondence toJayaraj Rajagopal.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Schematic representation of the dedifferentiation of luminal secretory cells into functional basal stem cells.

a, Differentiated luminal secretory cells are labelled with a YFP lineage tag in a homeostatic airway epithelium. Basal stem cells are then ablated using diphtheria toxin. In response, lineage-labelled secretory cells dedifferentiate into cells that morphologically resemble basal cells and express basal stem cell markers. These dedifferentiated basal-like cells respond to physiologically relevant toxic and infectious injury and serve as multipotent stem cells during epithelial regeneration. The inset depicts the different cell types of the airway epithelium. b, Graphical representation of the dedifferentiation potential of differentiated basal-like cells. x axis represents the maturity of a secretory cell; y axis represents the propensity for dedifferentiation to a basal-like cell. The propensity to dedifferentiate is inversely correlated to the maturity of the secretory cell.

Extended Data Figure 2 Inhaled doxycycline efficiently ablates basal stem cells of the airway epithelium.

a, Schematic representation of basal-cell-specific ablation using i-Dox. b, Co-labelling of p63 (green) and CK5 (red) on tracheal sections CK5-DTA mice that received either i-PBS (top) or i-Dox (middle and bottom panels show 2 and 3 doses of i-Dox, respectively). c, Co-labelling of NGFR (green) and T1α (red) i-PBS- or i-Dox-treated mice. d, Quantification of the number of p63+ (black bar) and CK5+ (grey bar) basal cells from CK5-DTA animals treated with PBS (p63, 1,229 ± 65.45; CK5, 1,376 ± 25.23), two doses of i-Dox (p63, 690 ± 35.13; CK5, 716 ± 12.44) or three doses of i-Dox (p63, 262 ± 29.5; CK5, 255 ± 46.82). y axis represents the absolute numbers of basal cells (from three independent tracheal sections). Dox(2) and Dox (3) refer to two and three doses of doxycycline inhalation, respectively. n = 3 (two mice per condition). Error bars, average ± s.e.m. Scale bars, 20 μm.

Extended Data Figure 3 Secretory cells begin to express proliferation and stem cell markers and undergo dedifferentiation following basal cell ablation.

a, Orthogonal confocal optical sections of SCGB1A1 (green), CK5 (cyan) and Ki67 (red) XY and XZ planes are shown to demonstrate the colocalization of Ki67 and SCGB1A1. b, Quantification of the percentage of Ki67+ CK8+ double-positive cells per total Ki67+ cells from i-PBS (23.74% ± 6.76)- and i-Dox (63.22% ± 4.14)-treated CK5-DTA mice. c, Co-labelling of CK5 and SCGB1A1 in i-Dox-treated CK5-DTA mice. White arrows, double-positive cells. d, Immunostaining for YFP (green) and Ki67 (red) on sections from SCGB1A1–YFP/CK5-DTA mice. White arrows indicate YFP+ Ki67+cells. e, Quantification of the percentage of YFP+Ki67+ cells per total Ki67+ cells in SCGB1A1–YFP/CK5-DTA mice that were treated with either i-Dox (31.74% ± 7.15) or i-PBS (9.65% ± 2.12). f, Co-labelling of YFP (green) with p63 or T1α (red) on tracheal sections of SCGB1A1–YFP/CK5-DTA mice that were either treated with i-PBS (top) or i-Dox (bottom). White arrows, double-positive cells. n = 3 (three mice per condition). Error bars, average ± s.e.m. Scale bars, 20 μm.

Extended Data Figure 4 Dissociation and fluorescence activated cell sorting of airway epithelial cells.

a, Schematic representation of tracheal epithelial cell dissociation from secretory cell lineage-labelled mice (SCGB1A1-CreER/LSL-YFP) after five doses of tamoxifen. Of the total epithelial cells, EPCAM+ CD24− cells were gated to remove ciliated cells. Then YFP+secretory cells and GSIβ4+ basal cells were sorted. b, Of the total epithelial cells, EPCAM+ CD24− cells were gated to remove ciliated cells (left). Then, YFP+ secretory cells were separated from GSIβ4+ basal cells (middle). Sorted YFP+ cells were also marked by the secretory cell marker SSEA-1 as expected for a pure population of SCG1A1+ cells (right).

Extended Data Figure 5 Sorted secretory cells dedifferentiate into basal-like self-renewing stem cells upon ex vivo culture.

a, Schematic representation of tracheal epithelial cell dissociation from basal cell reporter mice (CK5-rtTA/tet(O)H2BGFP) followed by sorting of GFP–SSEA-1+ secretory cells. Sorted SSEA-1+ cells were grown as spheres in Matrigel or plated on transwell membranes. Doxycycline was administered and cells were monitored for the initiation of GFP expression. b, Fluorescence-activated cell sorting of SSEA-1+ cells from basal cell reporter mice (CK5-rtTA/tet(O)H2BGFP). Arrows indicate gating windows. EPCAM is a pan-epithelial marker used to exclude non-epithelial lineages. Sorted SSEA-1+ secretory cells did not express GFP. c, Immunofluorescence staining for p63 (red; top) or CK5 (red; bottom) in combination with H2B–GFP (green; all panels) on sorted secretory cells that were either cultured as Matrigel spheres (left) or on transwells (right). Immunofluorescence analysis confirmed that H2B–GFP+ cells expressed p63 and CK5, again confirming that secretory cells dedifferentiate in culture. n = 3 (two replicates per condition). Scale bars, 20 μm.

Extended Data Figure 6 Sorted lineage-labelled secretory cells undergo dedifferentiation ex vivo, express basal stem cell markers, and can be serially passaged, as can secretory cells that underwent dedifferentiation in vivo.

a, Schematic representation of secretory cell labelling, sorting and subsequent culturing in Matrigel or on transwell membranes. b, Cell colonies obtained from early passage cultures of YFP+ secretory-cell-derived cells on transwell membranes. c, Immunostaining for CK5 (red), p63 (magenta) and YFP (green) on passage-five basal cell colonies from _ex vivo_-dedifferentiated cells. d, Schematic showing that YFP+ secretory-cell-derived spheres from SCGB1A1-CreER/LSL-YFP mice were serially passaged for five generations. e, Quantification of the sphere-forming efficiency: P1 (2.86% ± 0.65), P2 (3.36% ± 0.6), P3 (2.31% ± 0.32), P4 (2.75% ± 0.69) and P5 (2.7% ± 0.94). x axis, number of passages. y axis, percentage of spheres formed. f, Schematic representation of in vivo dedifferentiation followed by the sorting and culturing of YFP+ basal-like cells. g, Immunostaining for CK5 (red), p63 (magenta) and YFP (green) on passage-five cell colonies from _in vivo_-dedifferentiated cells. n = 3 (two replicates per condition). Error bars, average ± s.e.m. Scale bars, 20 μm.

Extended Data Figure 7 B1–eGFP transgenic mice express GFP in mature subsets of secretory cells.

a, Co-labelling of GFP (green) with CK5 or SCGB1A1 or SSEA-1 (all in red) on large airways sections derived from adult B1–eGFP transgenic mice at homeostasis. White arrows indicate SSEA-1+ B1–eGFP−, whereas white arrowheads point to cells that are B1–eGFP+ SSEA-1− (bottom). b, B1–eGFP trachea were stained for GFP (green) and SSEA-1 (red) on day 4 and day 6 after SO2-induced injury. n = 3 (two mice per condition per time point). Scale bars, 20 μm.

Extended Data Figure 8 Dedifferentiated basal-like stem cells are stable and self-renew to the same degree as endogenous basal stem cells.

a, Schematic representation of the dedifferentiation protocol to assess the ability of basal-like stem cells to persist and self-renew for 2 months. b, Immunofluorescence staining for YFP (green) in combination with CK5 (cyan) or Ki67 (red) on sections from SCGB1A1–YFP/CK5-DTA mice 2 months after basal cell ablation. c, Quantification of the percentage of proliferating dedifferentiated CK5+ YFP+ (10.16% ± 2.57) (green bar) and wild-type CK5+ YFP− (9.25% ± 0.79) (black bar) stem cells out of the total CK5+ stem cell population in the large airways of SCGB1A1–YFP/CK5-DTA mice 2 months after basal cell ablation. The white arrow points to a proliferating dedifferentiated basal-like stem cell. n = 3 (two mice per condition). Error bars, average ± s.e.m. Scale bar, 20 μm.

Extended Data Figure 9 SO2-and influenza-induced injury efficiently removes the suprabasal cells of the airway epithelium.

a, Immunofluorescence staining for CK5 (red) and YFP (green) in SCGB1A1–YFP/CK5-DTA airway epithelium 24 h after SO2 inhalation. Only a single layer of basal cells persists after injury. White arrows, CK5+ YFP+ double-positive cells (yellow). b, Immunofluorescence analysis of basal cells (CK5, red) and secretory cells (SCGB1A1, green) from uninjured (top), 72 hours post-infection (h.p.i.; middle) and 14 days post-infection (d.p.i.; bottom). White arrow, secretory cell debris (in green). n = 3 (two replicates per condition). Scale bars, 20 μm.

Extended Data Figure 10 _Ex vivo_-dedifferentiated cells can be clonally expanded and give rise to a complete airway epithelium in air–liquid interface cultures.

a, Schematic representation of the e_x vivo_ dedifferentiation of secretory cells, serial passage and differentiation. b, Whole-mount immunostaining for YFP (green) in combination with CK5 or p63 or acetylated (AC)-tubulin (red). c, Co-labelling of YFP (green) with CK5 or p63 or SCGB1A1 or acetylated-tubulin (red) on differentiated epithelium derived from serially passaged dedifferentiated basal-like cells. d, Whole-mount immunofluorescence staining for SCGB1A1 (red) and acetylated-tubulin (green) on clonally derived epithelium (left). Co-labelling of YFP (green) with CK5 (white) and CK8 (red; top) or SCGB1A1 and FOXJ1 (bottom) on cross sections of clonally derived epithelium. Scale bars, 20 μm.

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Tata, P., Mou, H., Pardo-Saganta, A. et al. Dedifferentiation of committed epithelial cells into stem cells in vivo .Nature 503, 218–223 (2013). https://doi.org/10.1038/nature12777

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