Single-cell gene profiling and lineage tracing analyses revealed novel mechanisms of endothelial repair by progenitors


Stem/progenitor cells (SPCs) have been implicated to participate in vascular repair. However, the exact role of SPCs in endothelial repair of large vessels still remains controversial. This study aimed to delineate the cellular heterogeneity and possible functional role of endogenous vascular SPCs in large vessels. Using single-cell RNA-sequencing (scRNA-seq) and genetic lineage tracing mouse models, we uncovered the cellular heterogeneity of SPCs, i.e., c-Kit+ cells in the mouse aorta, and found that endogenous c-Kit+ cells acquire endothelial cell fate in the aorta under both physiological and pathological conditions. While c-Kit+ cells contribute to aortic endothelial turnover in the atheroprone regions during homeostasis, recipient c-Kit+ cells of nonbone marrow source replace both luminal and microvessel endothelial cells in transplant arteriosclerosis. Single-cell pseudotime analysis of scRNA-seq data and in vitro cell experiments suggest that vascular SPCs display endothelial differentiation potential and undergo metabolic reprogramming during cell differentiation, in which AKT/mTOR-dependent glycolysis is critical for endothelial gene expression. These findings demonstrate a critical role for c-Kit lineage cells in aortic endothelial turnover and replacement, and may provide insights into therapeutic strategies for vascular diseases.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8





Endothelial cells


Extracellular acidification rate


Mesenchymal stromal cell


Natural killer


Oxygen consumption rate


Red blood cells


Stem cell antigen-1/lymphocyte antigen 6 complex, locus A


Single-cell RNA-sequencing


Smooth muscle cells


Stem/progenitor cells

TCA cycle:

Tricarboxylic acid cycle


Tandem dimer Tomato


Uniform manifold approximation and projection


Vascular endothelial growth factor


  1. 1.

    Zhang L, Issa Bhaloo S, Chen T, Zhou B, Xu Q (2018) Role of resident stem cells in vessel formation and arteriosclerosis. Circ Res 122(11):1608–1624.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  2. 2.

    Hu Y, Zhang Z, Torsney E, Afzal AR, Davison F, Metzler B, Xu Q (2004) Abundant progenitor cells in the adventitia contribute to atherosclerosis of vein grafts in ApoE-deficient mice. J Clin Invest 113(9):1258–1265.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  3. 3.

    Chen Y, Wong MM, Campagnolo P, Simpson R, Winkler B, Margariti A, Hu Y, Xu Q (2013) Adventitial stem cells in vein grafts display multilineage potential that contributes to neointimal formation. Arterioscler Thromb Vasc Biol 33(8):1844–1851.

    CAS  Article  PubMed  Google Scholar 

  4. 4.

    Fang S, Wei J, Pentinmikko N, Leinonen H, Salven P (2012) Generation of functional blood vessels from a single c-kit+ adult vascular endothelial stem cell. PLoS Biol 10(10):e1001407.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Psaltis PJ, Puranik AS, Spoon DB, Chue CD, Hoffman SJ, Witt TA, Delacroix S, Kleppe LS, Mueske CS, Pan S, Gulati R, Simari RD (2014) Characterization of a resident population of adventitial macrophage progenitor cells in postnatal vasculature. Circ Res 115(3):364–375.

    CAS  Article  PubMed  Google Scholar 

  6. 6.

    Mekala SR, Worsdorfer P, Bauer J, Stoll O, Wagner N, Reeh L, Loew K, Eckner G, Kwok CK, Wischmeyer E, Dickinson ME, Schulze H, Stegner D, Benndorf RA, Edenhofer F, Pfeiffer V, Kuerten S, Frantz S, Ergun S (2018) Generation of cardiomyocytes from vascular adventitia-resident stem cells. Circ Res 123(6):686–699.

    CAS  Article  PubMed  Google Scholar 

  7. 7.

    Grun D, van Oudenaarden A (2015) Design and analysis of single-cell sequencing experiments. Cell 163(4):799–810.

    CAS  Article  PubMed  Google Scholar 

  8. 8.

    Gu W, Ni Z, Tan YQ, Deng J, Zhang SJ, Lv ZC, Wang XJ, Chen T, Zhang Z, Hu Y, Jing ZC, Xu Q (2019) Adventitial cell atlas of wt (wild type) and ApoE (apolipoprotein E)-deficient mice defined by single-cell RNA sequencing. Arterioscler Thromb Vasc Biol 39(6):1055–1071.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Gu W, Nowak WN, Xie Y, Le Bras A, Hu Y, Deng J, Issa Bhaloo S, Lu Y, Yuan H, Fidanis E, Saxena A, Kanno T, Mason AJ, Dulak J, Cai J, Xu Q (2019) Single-cell RNA-sequencing and metabolomics analyses reveal the contribution of perivascular adipose tissue stem cells to vascular remodeling. Arterioscleros Thrombos Vasc Biol ATVBAHA119312732.

  10. 10.

    Kretzschmar K, Watt FM (2012) Lineage tracing. Cell 148(1–2):33–45.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. 11.

    van Berlo JH, Kanisicak O, Maillet M, Vagnozzi RJ, Karch J, Lin SC, Middleton RC, Marban E, Molkentin JD (2014) c-kit+ cells minimally contribute cardiomyocytes to the heart. Nature 509(7500):337–341.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Sultana N, Zhang L, Yan J, Chen J, Cai W, Razzaque S, Jeong D, Sheng W, Bu L, Xu M, Huang GY, Hajjar RJ, Zhou B, Moon A, Cai CL (2015) Resident c-kit(+) cells in the heart are not cardiac stem cells. Nat Commun 6:8701.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. 13.

    Liu Q, Yang R, Huang X, Zhang H, He L, Zhang L, Tian X, Nie Y, Hu S, Yan Y, Zhang L, Qiao Z, Wang QD, Lui KO, Zhou B (2016) Genetic lineage tracing identifies in situ Kit-expressing cardiomyocytes. Cell Res 26(1):119–130.

    CAS  Article  PubMed  Google Scholar 

  14. 14.

    Vagnozzi RJ, Sargent MA, Lin SJ, Palpant NJ, Murry CE, Molkentin JD (2018) Genetic lineage tracing of Sca-1(+) cells reveals endothelial but not myogenic contribution to the murine heart. Circulation 138(25):2931–2939.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  15. 15.

    Tang J, Li Y, Huang X, He L, Zhang L, Wang H, Yu W, Pu W, Tian X, Nie Y, Hu S, Wang QD, Lui KO, Zhou B (2018) Fate mapping of Sca1(+) cardiac progenitor cells in the adult mouse heart. Circulation 138(25):2967–2969.

    CAS  Article  PubMed  Google Scholar 

  16. 16.

    Neidig LE, Weinberger F, Palpant NJ, Mignone J, Martinson AM, Sorensen DW, Bender I, Nemoto N, Reinecke H, Pabon L, Molkentin JD, Murry CE, van Berlo JH (2018) Evidence for minimal cardiogenic potential of stem cell antigen 1-positive cells in the adult mouse heart. Circulation 138(25):2960–2962.

    Article  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Zhang L, Sultana N, Yan J, Yang F, Chen F, Chepurko E, Yang FC, Du Q, Zangi L, Xu M, Bu L, Cai CL (2018) Cardiac Sca-1(+) cells are not intrinsic stem cells for myocardial development, renewal, and repair. Circulation 138(25):2919–2930.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  18. 18.

    Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13(1):133–140.

    CAS  Article  Google Scholar 

  19. 19.

    Liu Q, Huang X, Zhang H, Tian X, He L, Yang R, Yan Y, Wang QD, Gillich A, Zhou B (2015) c-kit(+) cells adopt vascular endothelial but not epithelial cell fates during lung maintenance and repair. Nat Med 21(8):866–868.

    CAS  Article  PubMed  Google Scholar 

  20. 20.

    Robinet P, Milewicz DM, Cassis LA, Leeper NJ, Lu HS, Smith JD (2018) Consideration of sex differences in design and reporting of experimental arterial pathology studies-statement from ATVB council. Arterioscler Thromb Vasc Biol 38(2):292–303.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Dietrich H, Hu Y, Zou Y, Dirnhofer S, Kleindienst R, Wick G, Xu Q (2000) Mouse model of transplant arteriosclerosis: role of intercellular adhesion molecule-1. Arterioscler Thromb Vasc Biol 20(2):343–352.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Hu Y, Davison F, Ludewig B, Erdel M, Mayr M, Url M, Dietrich H, Xu Q (2002) Smooth muscle cells in transplant atherosclerotic lesions are originated from recipients, but not bone marrow progenitor cells. Circulation 106(14):1834–1839.

    Article  PubMed  Google Scholar 

  23. 23.

    Butler A, Hoffman P, Smibert P, Papalexi E, Satija R (2018) Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 36(5):411–420.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  24. 24.

    Dobnikar L, Taylor AL, Chappell J, Oldach P, Harman JL, Oerton E, Dzierzak E, Bennett MR, Spivakov M, Jorgensen HF (2018) Disease-relevant transcriptional signatures identified in individual smooth muscle cells from healthy mouse vessels. Nat Commun 9(1):4567.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. 25.

    McDonald AI, Shirali AS, Aragon R, Ma F, Hernandez G, Vaughn DA, Mack JJ, Lim TY, Sunshine H, Zhao P, Kalinichenko V, Hai T, Pelegrini M, Ardehali R, Iruela-Arispe ML (2018) Endothelial regeneration of large vessels is a biphasic process driven by local cells with distinct proliferative capacities. Cell Stem Cell 23(2):210–225.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Tabula Muris C, Overall c, Logistical c, Organ c, processing, Library p, sequencing, Computational data a, Cell type a, Writing g, Supplemental text writing g, Principal i (2018) Single-cell transcriptomics of 20 mouse organs creates a Tabula Muris. Nature 562(7727):367–372.

  27. 27.

    Kalluri AS, Vellarikkal SK, Edelman ER, Nguyen L, Subramanian A, Ellinor PT, Regev A, Kathiresan S, Gupta RM (2019) Single cell analysis of the normal mouse aorta reveals functionally distinct endothelial cell populations. Circulation.

    Article  PubMed  PubMed Central  Google Scholar 

  28. 28.

    Qiu X, Mao Q, Tang Y, Wang L, Chawla R, Pliner HA, Trapnell C (2017) Reversed graph embedding resolves complex single-cell trajectories. Nat Methods 14(10):979–982.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. 29.

    Durinck S, Spellman PT, Birney E, Huber W (2009) Mapping identifiers for the integration of genomic datasets with the R/Bioconductor package biomaRt. Nat Protoc 4(8):1184–1191.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. 30.

    da Huang W, Sherman BT, Lempicki RA (2009) Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 4(1):44–57.

    CAS  Article  Google Scholar 

  31. 31.

    Wang D, Li LK, Dai T, Wang A, Li S (2018) Adult stem cells in vascular remodeling. Theranostics 8(3):815–829.

    Article  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Lennartsson J, Ronnstrand L (2012) Stem cell factor receptor/c-Kit: from basic science to clinical implications. Physiol Rev 92(4):1619–1649.

    CAS  Article  PubMed  Google Scholar 

  33. 33.

    Foteinos G, Hu Y, Xiao Q, Metzler B, Xu Q (2008) Rapid endothelial turnover in atherosclerosis-prone areas coincides with stem cell repair in apolipoprotein E-deficient mice. Circulation 117(14):1856–1863.

    Article  PubMed  Google Scholar 

  34. 34.

    Chen Q, Yang M, Wu H, Zhou J, Wang W, Zhang H, Zhao L, Zhu J, Zhou B, Xu Q, Zhang L (2018) Genetic lineage tracing analysis of c-kit(+) stem/progenitor cells revealed a contribution to vascular injury-induced neointimal lesions. J Mol Cell Cardiol 121:277–286.

    CAS  Article  PubMed  Google Scholar 

  35. 35.

    Roostalu U, Aldeiri B, Albertini A, Humphreys N, Simonsen-Jackson M, Wong JKF, Cossu G (2018) Distinct cellular mechanisms underlie smooth muscle turnover in vascular development and repair. Circ Res 122(2):267–281.

    CAS  Article  PubMed  Google Scholar 

  36. 36.

    Ni Z, Deng J, Potter CMF, Nowak WN, Gu W, Zhang Z, Chen T, Chen Q, Hu Y, Zhou B, Xu Q, Zhang L (2019) Recipient c-Kit lineage cells repopulate smooth muscle cells of transplant arteriosclerosis in mouse models. Circ Res 125(2):223–241.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Hu YH, Davison F, Zhang ZY, Xu QB (2003) Endothelial replacement and angiogenesis in arteriosclerotic lesions of allografts are contributed by circulating progenitor cells. Circulation 108(25):3122–3127.

    Article  PubMed  Google Scholar 

  38. 38.

    Shi CW, Russell ME, Bianchi C, Newell JB, Haber E (1994) Murine model of accelerated transplant arteriosclerosis. Circ Res 75(2):199–207.

    CAS  Article  PubMed  Google Scholar 

  39. 39.

    Chow LH, Huh S, Jiang J, Zhong R, Pickering JG (1996) Intimal thickening develops without humoral immunity in a mouse aortic allograft model of chronic vascular rejection. Circulation 94(12):3079–3082

    CAS  Article  Google Scholar 

  40. 40.

    Eelen G, de Zeeuw P, Treps L, Harjes U, Wong BW, Carmeliet P (2018) Endothelial cell metabolism. Physiol Rev 98(1):3–58.

    CAS  Article  PubMed  Google Scholar 

  41. 41.

    Karar J, Maity A (2011) PI3K/AKT/mTOR pathway in angiogenesis. Front Mol Neurosci 4:51.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Singec I, Jandial R, Crain A, Nikkhah G, Snyder EY (2007) The leading edge of stem cell therapeutics. Annu Rev Med 58:313–328.

    CAS  Article  PubMed  Google Scholar 

  43. 43.

    Kipshidze N, Dangas G, Tsapenko M, Moses J, Leon MB, Kutryk M, Serruys P (2004) Role of the endothelium in modulating neointimal formation: vasculoprotective approaches to attenuate restenosis after percutaneous coronary interventions. J Am Coll Cardiol 44(4):733–739.

    CAS  Article  PubMed  Google Scholar 

  44. 44.

    Sedding DG, Boyle EC, Demandt JAF, Sluimer JC, Dutzmann J, Haverich A, Bauersachs J (2018) Vasa vasorum angiogenesis: key player in the initiation and progression of atherosclerosis and potential target for the treatment of cardiovascular disease. Front Immunol 9:706.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Kusumbe AP, Ramasamy SK, Adams RH (2014) Coupling of angiogenesis and osteogenesis by a specific vessel subtype in bone. Nature 507(7492):323–328.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. 46.

    Yu QC, Song W, Wang D, Zeng YA (2016) Identification of blood vascular endothelial stem cells by the expression of protein C receptor. Cell Res 26(10):1079–1098.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  47. 47.

    Wakabayashi T, Naito H, Suehiro JI, Lin Y, Kawaji H, Iba T, Kouno T, Ishikawa-Kato S, Furuno M, Takara K, Muramatsu F, Weizhen J, Kidoya H, Ishihara K, Hayashizaki Y, Nishida K, Yoder MC, Takakura N (2018) CD157 Marks tissue-resident endothelial stem cells with homeostatic and regenerative properties. Cell Stem Cell 22(3):384–397.

    CAS  Article  PubMed  Google Scholar 

  48. 48.

    Patel J, Seppanen EJ, Rodero MP, Wong HY, Donovan P, Neufeld Z, Fisk NM, Francois M, Khosrotehrani K (2017) Functional definition of progenitors versus mature endothelial cells reveals key SoxF-dependent differentiation process. Circulation 135(8):786–805.

    CAS  Article  PubMed  Google Scholar 

  49. 49.

    Ingram DA, Mead LE, Moore DB, Woodard W, Fenoglio A, Yoder MC (2005) Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells. Blood 105(7):2783–2786.

    CAS  Article  PubMed  Google Scholar 

  50. 50.

    Naito H, Kidoya H, Sakimoto S, Wakabayashi T, Takakura N (2012) Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels. EMBO J 31(4):842–855.

    CAS  Article  PubMed  Google Scholar 

  51. 51.

    Woodfin A, Voisin MB, Nourshargh S (2007) PECAM-1: a multi-functional molecule in inflammation and vascular biology. Arterioscler Thromb Vasc Biol 27(12):2514–2523.

    CAS  Article  PubMed  Google Scholar 

  52. 52.

    Purhonen S, Palm J, Rossi D, Kaskenpaa N, Rajantie I, Yla-Herttuala S, Alitalo K, Weissman IL, Salven P (2008) Bone marrow-derived circulating endothelial precursors do not contribute to vascular endothelium and are not needed for tumor growth. Proc Natl Acad Sci USA 105(18):6620–6625.

    Article  PubMed  Google Scholar 

  53. 53.

    Hagensen MK, Shim J, Falk E, Bentzon JF (2011) Flanking recipient vasculature, not circulating progenitor cells, contributes to endothelium and smooth muscle in murine allograft vasculopathy. Arterioscler Thromb Vasc Biol 31(4):808–813.

    CAS  Article  PubMed  Google Scholar 

  54. 54.

    Kamran P, Sereti KI, Zhao P, Ali SR, Weissman IL, Ardehali R (2013) Parabiosis in mice: a detailed protocol. J Vis Exp 2013:80.

    CAS  Article  Google Scholar 

  55. 55.

    Rudd JH, Warburton EA, Fryer TD, Jones HA, Clark JC, Antoun N, Johnstrom P, Davenport AP, Kirkpatrick PJ, Arch BN, Pickard JD, Weissberg PL (2002) Imaging atherosclerotic plaque inflammation with [18F]-fluorodeoxyglucose positron emission tomography. Circulation 105(23):2708–2711

    CAS  Article  Google Scholar 

  56. 56.

    Hall JL, Chatham JC, Eldar-Finkelman H, Gibbons GH (2001) Upregulation of glucose metabolism during intimal lesion formation is coupled to the inhibition of vascular smooth muscle cell apoptosis. Role of GSK3beta. Diabetes 50(5):1171–1179

    CAS  Article  Google Scholar 

  57. 57.

    Tomas L, Edsfeldt A, Mollet IG, Perisic Matic L, Prehn C, Adamski J, Paulsson-Berne G, Hedin U, Nilsson J, Bengtsson E, Goncalves I, Bjorkbacka H (2018) Altered metabolism distinguishes high-risk from stable carotid atherosclerotic plaques. Eur Heart J 39(24):2301–2310.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. 58.

    Folmes CD, Dzeja PP, Nelson TJ, Terzic A (2012) Metabolic plasticity in stem cell homeostasis and differentiation. Cell Stem Cell 11(5):596–606.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  59. 59.

    Lai L, Reineke E, Hamilton DJ, Cooke JP (2019) Glycolytic switch is required for transdifferentiation to endothelial lineage. Circulation 139(1):119–133.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Rohlenova K, Veys K, Miranda-Santos I, De Bock K, Carmeliet P (2018) Endothelial cell metabolism in health and disease. Trends Cell Biol 28(3):224–236.

    CAS  Article  PubMed  Google Scholar 

  61. 61.

    He L, Li Y, Li Y, Pu W, Huang X, Tian X, Wang Y, Zhang H, Liu Q, Zhang L, Zhao H, Tang J, Ji H, Cai D, Han Z, Han Z, Nie Y, Hu S, Wang QD, Sun R, Fei J, Wang F, Chen T, Yan Y, Huang H, Pu WT, Zhou B (2017) Enhancing the precision of genetic lineage tracing using dual recombinases. Nat Med 23(12):1488–1498.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  62. 62.

    Leeper NJ, Hunter AL, Cooke JP (2010) Stem cell therapy for vascular regeneration: adult, embryonic, and induced pluripotent stem cells. Circulation 122(5):517–526.

    Article  PubMed  PubMed Central  Google Scholar 

Download references


This work was supported by grants from British Heart Foundation (RG/14/6/31144), National Natural Science Foundation of China (81220108004, 81570249, 81930010, 81870206, 91339102, 91639302, 91539103, and 31830039), Zhejiang Provincial Natural Science Foundation (LD18H020001) and Royal Society-Newton Advanced Fellowship (NA170109). Some panels in the figure were produced using Servier Medical Art under a Creative Commons Attribution 3.0 Unported License.

Author information



Corresponding authors

Correspondence to Li Zhang or Qingbo Xu.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Deng, J., Ni, Z., Gu, W. et al. Single-cell gene profiling and lineage tracing analyses revealed novel mechanisms of endothelial repair by progenitors. Cell. Mol. Life Sci. 77, 5299–5320 (2020).

Download citation


  • Endothelial repair
  • Lineage tracing
  • Metabolism
  • Single-cell RNA-sequencing
  • Stem cells