Molecular and Cellular Biochemistry

, Volume 392, Issue 1–2, pp 187–204 | Cite as

Human mesenchymal stem cells express a myofibroblastic phenotype in vitro: comparison to human cardiac myofibroblasts

  • Melanie A. Ngo
  • Alison Müller
  • Yun Li
  • Shannon Neumann
  • Ganghong Tian
  • Ian M. C. Dixon
  • Rakesh C. Arora
  • Darren H. FreedEmail author


Cardiac fibrosis accompanies a variety of myocardial disorders, and is induced by myofibroblasts. These cells may be composed of a heterogeneous population of parent cells, including interstitial fibroblasts and circulating progenitor cells. Direct comparison of human bone marrow-derived mesenchymal stem cells (BM-MSCs) and cardiac myofibroblasts (CMyfbs) has not been previously reported. We hypothesized that BM-MSCs readily adopt a myofibroblastic phenotype in culture. Human primary BM-MSCs and human CMyfbs were isolated from patients undergoing open heart surgery and expanded under standard culture conditions. We assessed and compared their phenotypic and functional characteristics by examining their gene expression profile, their ability to contract collagen gels and synthesize collagen type I. In addition, we examined the role of non-muscle myosin II (NMMII) in modulating MSC myogenic function using NMMII siRNA knockdown and blebbistatin, a specific small molecule inhibitor of NMMII. We report that, while human BM-MSCs retain pluripotency, they adopt a myofibroblastic phenotype in culture and stain positive for the myofibroblast markers α-SMA, vimentin, NMMIIB, ED-A fibronectin, and collagen type 1 at each passage. In addition, they contract collagen gels in response to TGF-β1 and synthesize collagen similar to human CMyfbs. Moreover, inhibition of NMMII activity with blebbistatin completely attenuates gel contractility without affecting cell viability. Thus, human BM-MSCs share and exhibit similar physiological and functional characteristics as human CMyfbs in vitro, and their propensity to adopt a myofibroblast phenotype in culture may contribute to cardiac fibrosis.


Cardiac fibrosis Stem cell differentiation Myofibroblast contractility Blebbistatin Cellular contraction Wound healing 



We would like to thank Ryan H. Cunnington, Aresh Sepehri, and Steve Wayne for their technical assistance and loans of equipment. This work was supported by funding from the Canadian Institutes for Health Research (New Emerging Team Grant to GT, RCA and DHF; Operating Grant to IMCD), the Heart and Stroke Foundation (grant-in-aid to IMCD) and the St. Boniface Hospital Foundation.

Conflict of interest



  1. 1.
    van den Borne SW, Diez J, Blankesteijn WM, Verjans J, Hofstra L, Narula J (2010) Myocardial remodeling after infarction: the role of myofibroblasts. Nat Rev Cardiol 7(1):30–37PubMedCrossRefGoogle Scholar
  2. 2.
    Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and mechano-regulation of connective tissue remodelling. Nat Rev Mol Cell Biol 3(5):349–363PubMedCrossRefGoogle Scholar
  3. 3.
    van Amerongen MJ, Bou-Gharios G, Popa E, van Ark J, Petersen AH, van Dam GM, van Luyn MJ, Harmsen MC (2008) Bone marrow-derived myofibroblasts contribute functionally to scar formation after myocardial infarction. J Pathol 214(3):377–386PubMedCrossRefGoogle Scholar
  4. 4.
    Mollmann H, Nef HM, Kostin S, von KC, Pilz I, Weber M, Schaper J, Hamm CW, Elsasser A (2006) Bone marrow-derived cells contribute to infarct remodelling. Cardiovasc Res 71(4):661–671PubMedCrossRefGoogle Scholar
  5. 5.
    Haudek SB, Xia Y, Huebener P, Lee JM, Carlson S, Crawford JR, Pilling D, Gomer RH, Trial J, Frangogiannis NG, Entman ML (2006) Bone marrow-derived fibroblast precursors mediate ischemic cardiomyopathy in mice. Proc Natl Acad Sci USA 103(48):18284–18289PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Chang HY, Chi JT, Dudoit S, Bondre C, van de Rijn M, Botstein D, Brown PO (2002) Diversity, topographic differentiation, and positional memory in human fibroblasts. Proc Natl Acad Sci USA 99(20):12877–12882PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Kania G, Blyszczuk P, Stein S, Valaperti A, Germano D, Dirnhofer S, Hunziker L, Matter CM, Eriksson U (2009) Heart-infiltrating prominin-1+/CD133+ progenitor cells represent the cellular source of transforming growth factor β-mediated cardiac fibrosis in experimental autoimmune myocarditis. Circ Res 105(5):462–470PubMedCrossRefGoogle Scholar
  8. 8.
    Sopel MJ, Rosin NL, Lee TD, Legare JF (2011) Myocardial fibrosis in response to angiotensin II is preceded by the recruitment of mesenchymal progenitor cells. Lab Invest 91(4):565–578PubMedCrossRefGoogle Scholar
  9. 9.
    McBeath R, Pirone DM, Nelson CM, Bhadriraju K, Chen CS (2004) Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Dev Cell 6(4):483–495PubMedCrossRefGoogle Scholar
  10. 10.
    Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143–147PubMedCrossRefGoogle Scholar
  11. 11.
    Woodbury D, Schwarz EJ, Prockop DJ, Black IB (2000) Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 61(4):364–370PubMedCrossRefGoogle Scholar
  12. 12.
    Engler AJ, Sen S, Sweeney HL, Discher DE (2006) Matrix elasticity directs stem cell lineage specification. Cell 126(4):677–689PubMedCrossRefGoogle Scholar
  13. 13.
    Bond JE, Ho TQ, Selim MA, Hunter CL, Bowers EV, Levinson H (2011) Temporal spatial expression and function of non-muscle myosin II isoforms IIA and IIB in scar remodeling. Lab Invest 91(4):499–508PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Wang N, Tolic-Norrelykke IM, Chen J, Mijailovich SM, Butler JP, Fredberg JJ, Stamenovic D (2002) Cell prestress. I. Stiffness and prestress are closely associated in adherent contractile cells. Am J Physiol Cell Physiol 282(3):C606–C616PubMedCrossRefGoogle Scholar
  15. 15.
    Even-Ram S, Doyle AD, Conti MA, Matsumoto K, Adelstein RS, Yamada KM (2007) Myosin IIA regulates cell motility and actomyosin–microtubule crosstalk. Nat Cell Biol 9(3):299–309PubMedCrossRefGoogle Scholar
  16. 16.
    Meshel AS, Wei Q, Adelstein RS, Sheetz MP (2005) Basic mechanism of three-dimensional collagen fibre transport by fibroblasts. Nat Cell Biol 7(2):157–164PubMedCrossRefGoogle Scholar
  17. 17.
    Abe M, Ho CH, Kamm KE, Grinnell F (2003) Different molecular motors mediate platelet-derived growth factor and lysophosphatidic acid-stimulated floating collagen matrix contraction. J Biol Chem 278(48):47707–47712PubMedCrossRefGoogle Scholar
  18. 18.
    Vicente-Manzanares M, Ma X, Adelstein RS, Horwitz AR (2009) Non-muscle myosin II takes centre stage in cell adhesion and migration. Nat Rev Mol Cell Biol 10(11):778–790PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Flynn PG, Helfman DM (2010) Non-muscle myosin IIB helps mediate TNF cell death signaling independent of actomyosin contractility (AMC). J Cell Biochem 110(6):1365–1375PubMedCrossRefGoogle Scholar
  20. 20.
    Kovacs M, Wang F, Hu A, Zhang Y, Sellers JR (2003) Functional divergence of human cytoplasmic myosin II: kinetic characterization of the non-muscle IIA isoform. J Biol Chem 278(40):38132–38140PubMedCrossRefGoogle Scholar
  21. 21.
    Conti MA, Even-Ram S, Liu C, Yamada KM, Adelstein RS (2004) Defects in cell adhesion and the visceral endoderm following ablation of nonmuscle myosin heavy chain II-A in mice. J Biol Chem 279(40):41263–41266PubMedCrossRefGoogle Scholar
  22. 22.
    Walker A, Su H, Conti MA, Harb N, Adelstein RS, Sato N (2010) Non-muscle myosin II regulates survival threshold of pluripotent stem cells. Nat Commun 1(6). doi: 10.1038/ncomms1074
  23. 23.
    Chen G, Hou Z, Gulbranson DR, Thomson JA (2010) Actin–myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells. Cell Stem Cell 7(2):240–248PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Ohgushi M, Matsumura M, Eiraku M, Murakami K, Aramaki T, Nishiyama A, Muguruma K, Nakano T, Suga H, Ueno M, Ishizaki T, Suemori H, Narumiya S, Niwa H, Sasai Y (2010) Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells. Cell Stem Cell 7(2):225–239PubMedCrossRefGoogle Scholar
  25. 25.
    Limouze J, Straight AF, Mitchison T, Sellers JR (2004) Specificity of blebbistatin, an inhibitor of myosin II. J Muscle Res Cell Motil 25(4–5):337–341PubMedCrossRefGoogle Scholar
  26. 26.
    Friedenstein AJ, Gorskaja JF, Kulagina NN (1976) Fibroblast precursors in normal and irradiated mouse hematopoietic organs. Exp Hematol 4(5):267–274PubMedGoogle Scholar
  27. 27.
    Covas DT, Panepucci RA, Fontes AM, Silva WA Jr, Orellana MD, Freitas MC, Neder L, Santos AR, Peres LC, Jamur MC, Zago MA (2008) Multipotent mesenchymal stromal cells obtained from diverse human tissues share functional properties and gene-expression profile with CD146 + perivascular cells and fibroblasts. Exp Hematol 36(5):642–654PubMedCrossRefGoogle Scholar
  28. 28.
    Bianco P, Robey PG, Simmons PJ (2008) Mesenchymal stem cells: revisiting history, concepts, and assays. Cell Stem Cell 2(4):313–319PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Wagner W, Ho AD (2007) Mesenchymal stem cell preparations—comparing apples and oranges. Stem Cell Rev 3(4):239–248PubMedCrossRefGoogle Scholar
  30. 30.
    Ball SG, Shuttleworth AC, Kielty CM (2004) Direct cell contact influences bone marrow mesenchymal stem cell fate. Int J Biochem Cell Biol 36(4):714–727PubMedCrossRefGoogle Scholar
  31. 31.
    Traverse JH, Henry TD, Pepine CJ, Willerson JT, Zhao DX, Ellis SG, Forder JR, Anderson RD, Hatzopoulos AK, Penn MS, Perin EC, Chambers J, Baran KW, Raveendran G, Lambert C, Lerman A, Simon DI, Vaughan DE, Lai D, Gee AP, Taylor DA, Cogle CR, Thomas JD, Olson RE, Bowman S, Francescon J, Geither C, Handberg E, Kappenman C, Westbrook L, Piller LB, Simpson LM, Baraniuk S, Loghin C, Aguilar D, Richman S, Zierold C, Spoon DB, Bettencourt J, Sayre SL, Vojvodic RW, Skarlatos SI, Gordon DJ, Ebert RF, Kwak M, Moye LA, Simari RD (2012) Effect of the use and timing of bone marrow mononuclear cell delivery on left ventricular function after acute myocardial infarction: the TIME randomized trial. J Am Med Assoc 308(22):2380–2389CrossRefGoogle Scholar
  32. 32.
    Perin EC, Willerson JT, Pepine CJ, Henry TD, Ellis SG, Zhao DX, Silva GV, Lai D, Thomas JD, Kronenberg MW, Martin AD, Anderson RD, Traverse JH, Penn MS, Anwaruddin S, Hatzopoulos AK, Gee AP, Taylor DA, Cogle CR, Smith D, Westbrook L, Chen J, Handberg E, Olson RE, Geither C, Bowman S, Francescon J, Baraniuk S, Piller LB, Simpson LM, Loghin C, Aguilar D, Richman S, Zierold C, Bettencourt J, Sayre SL, Vojvodic RW, Skarlatos SI, Gordon DJ, Ebert RF, Kwak M, Moye LA, Simari RD (2012) Effect of transendocardial delivery of autologous bone marrow mononuclear cells on functional capacity, left ventricular function, and perfusion in chronic heart failure: the FOCUS-CCTRN trial. J Am Med Assoc 307(16):1717–1726CrossRefGoogle Scholar
  33. 33.
    Traverse JH, Henry TD, Ellis SG, Pepine CJ, Willerson JT, Zhao DX, Forder JR, Byrne BJ, Hatzopoulos AK, Penn MS, Perin EC, Baran KW, Chambers J, Lambert C, Raveendran G, Simon DI, Vaughan DE, Simpson LM, Gee AP, Taylor DA, Cogle CR, Thomas JD, Silva GV, Jorgenson BC, Olson RE, Bowman S, Francescon J, Geither C, Handberg E, Smith DX, Baraniuk S, Piller LB, Loghin C, Aguilar D, Richman S, Zierold C, Bettencourt J, Sayre SL, Vojvodic RW, Skarlatos SI, Gordon DJ, Ebert RF, Kwak M, Moye LA, Simari RD (2011) Effect of intracoronary delivery of autologous bone marrow mononuclear cells 2 to 3 weeks following acute myocardial infarction on left ventricular function: the LateTIME randomized trial. J Am Med Assoc 306(19):2110–2119CrossRefGoogle Scholar
  34. 34.
    Caplan AI (1991) Mesenchymal stem cells. J Orthop Res 9(5):641–650PubMedCrossRefGoogle Scholar
  35. 35.
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85PubMedCrossRefGoogle Scholar
  36. 36.
    Pittenger MF, Mosca JD, McIntosh KR (2000) Human mesenchymal stem cells: progenitor cells for cartilage, bone, fat and stroma. Curr Top Microbiol Immunol 251:3–11PubMedGoogle Scholar
  37. 37.
    Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop D, Horwitz E (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315–317PubMedCrossRefGoogle Scholar
  38. 38.
    Santiago JJ, Dangerfield AL, Rattan SG, Bathe KL, Cunnington RH, Raizman JE, Bedosky KM, Freed DH, Kardami E, Dixon IM (2010) Cardiac fibroblast to myofibroblast differentiation in vivo and in vitro: expression of focal adhesion components in neonatal and adult rat ventricular myofibroblasts. Dev Dyn 239(6):1573–1584PubMedCrossRefGoogle Scholar
  39. 39.
    Chilton L, Ohya S, Freed D, George E, Drobic V, Shibukawa Y, Maccannell KA, Imaizumi Y, Clark RB, Dixon IM, Giles WR (2005) K + currents regulate the resting membrane potential, proliferation, and contractile responses in ventricular fibroblasts and myofibroblasts. Am J Physiol Heart Circ Physiol 288(6):H2931–H2939PubMedCrossRefGoogle Scholar
  40. 40.
    Drobic V, Cunnington RH, Bedosky KM, Raizman JE, Elimban VV, Rattan SG, Dixon IM (2007) Differential and combined effects of cardiotrophin-1 and TGF-beta1 on cardiac myofibroblast proliferation and contraction. Am J Physiol Heart Circ Physiol 293(2):H1053–H1064PubMedCrossRefGoogle Scholar
  41. 41.
    Cunnington RH, Wang B, Ghavami S, Bathe KL, Rattan SG, Dixon IM (2011) Antifibrotic properties of c-Ski and its regulation of cardiac myofibroblast phenotype and contractility. Am J Physiol Cell Physiol 300(1):C176–C186PubMedCrossRefGoogle Scholar
  42. 42.
    Prockop DJ, Kivirikko KI (1995) Collagens: molecular biology, diseases, and potentials for therapy. Ann Rev Biochem 64:403–434PubMedCrossRefGoogle Scholar
  43. 43.
    Altman GH, Horan RL, Martin I, Farhadi J, Stark PR, Volloch V, Richmond JC, Vunjak-Novakovic G, Kaplan DL (2002) Cell differentiation by mechanical stress. FASEB 16(2):270–272Google Scholar
  44. 44.
    Noth U, Schupp K, Heymer A, Kall S, Jakob F, Schutze N, Baumann B, Barthel T, Eulert J, Hendrich C (2005) Anterior cruciate ligament constructs fabricated from human mesenchymal stem cells in a collagen type I hydrogel. Cytotherapy 7(5):447–455PubMedCrossRefGoogle Scholar
  45. 45.
    Moreau JE, Chen J, Bramono DS, Volloch V, Chernoff H, Vunjak-Novakovic G, Richmond JC, Kaplan DL, Altman GH (2005) Growth factor induced fibroblast differentiation from human bone marrow stromal cells in vitro. J Orthop Res 23(1):164–174PubMedCrossRefGoogle Scholar
  46. 46.
    Hankemeier S, Keus M, Zeichen J, Jagodzinski M, Barkhausen T, Bosch U, Krettek C, Van Griensven M (2005) Modulation of proliferation and differentiation of human bone marrow stromal cells by fibroblast growth factor 2: potential implications for tissue engineering of tendons and ligaments. Tissue Eng 11(1–2):41–49PubMedCrossRefGoogle Scholar
  47. 47.
    Lee CH, Shah B, Moioli EK, Mao JJ (2010) CTGF directs fibroblast differentiation from human mesenchymal stem/stromal cells and defines connective tissue healing in a rodent injury model. J Clin Invest 120(9):3340–3349PubMedCentralPubMedCrossRefGoogle Scholar
  48. 48.
    Tse JR, Engler AJ (2011) Stiffness gradients mimicking in vivo tissue variation regulate mesenchymal stem cell fate. PLoS ONE 6(1):e15978PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Schallmoser K, Bartmann C, Rohde E, Bork S, Guelly C, Obenauf AC, Reinisch A, Horn P, Ho AD, Strunk D, Wagner W (2010) Replicative senescence-associated gene expression changes in mesenchymal stromal cells are similar under different culture conditions. Haematologica 95(6):867–874PubMedCentralPubMedCrossRefGoogle Scholar
  50. 50.
    Pevsner-Fischer M, Levin S, Zipori D (2011) The origins of mesenchymal stromal cell heterogeneity. Stem Cell Rev 7(3):560–568PubMedCrossRefGoogle Scholar
  51. 51.
    Roberts AB, Sporn MB, Assoian RK, Smith JM, Roche NS, Wakefield LM, Heine UI, Liotta LA, Falanga V, Kehrl JH et al (1986) Transforming growth factor type beta: rapid induction of fibrosis and angiogenesis in vivo and stimulation of collagen formation in vitro. PNAS 83(12):4167–4171PubMedCentralPubMedCrossRefGoogle Scholar
  52. 52.
    Kinner B, Zaleskas JM, Spector M (2002) Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells. Exp Cell Res 278(1):72–83PubMedCrossRefGoogle Scholar
  53. 53.
    Cieslik KA, Trial J, Entman ML (2011) Defective myofibroblast formation from mesenchymal stem cells in the aging murine heart rescue by activation of the AMPK pathway. Am J Pathol 179(4):1792–1806PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Desmouliere A, Geinoz A, Gabbiani F, Gabbiani G (1993) Transforming growth factor-beta 1 induces alpha-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts. J Cell Biol 122(1):103–111PubMedCrossRefGoogle Scholar
  55. 55.
    Jarnagin WR, Rockey DC, Koteliansky VE, Wang SS, Bissell DM (1994) Expression of variant fibronectins in wound healing: cellular source and biological activity of the EIIIA segment in rat hepatic fibrogenesis. J Cell Biol 127(6 Pt 2):2037–2048PubMedCrossRefGoogle Scholar
  56. 56.
    Serini G, Bochaton-Piallat ML, Ropraz P, Geinoz A, Borsi L, Zardi L, Gabbiani G (1998) The fibronectin domain ED-A is crucial for myofibroblastic phenotype induction by transforming growth factor-beta1. J Cell Biol 142(3):873–881PubMedCentralPubMedCrossRefGoogle Scholar
  57. 57.
    van der Straaten HM, Canninga-van Dijk MR, Verdonck LF, Castigliego D, Borst HP, Aten J, Fijnheer R (2004) Extra-domain-A fibronectin: a new marker of fibrosis in cutaneous graft-versus-host disease. J Invest Dermatol 123(6):1057–1062PubMedCrossRefGoogle Scholar
  58. 58.
    Sarugaser R, Hanoun L, Keating A, Stanford WL, Davies JE (2009) Human mesenchymal stem cells self-renew and differentiate according to a deterministic hierarchy. PLoS ONE 4(8):e6498PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Melanie A. Ngo
    • 1
  • Alison Müller
    • 1
  • Yun Li
    • 1
  • Shannon Neumann
    • 1
  • Ganghong Tian
    • 3
  • Ian M. C. Dixon
    • 1
  • Rakesh C. Arora
    • 1
    • 2
    • 3
  • Darren H. Freed
    • 1
    • 2
    • 3
    • 4
    Email author
  1. 1.Department of Physiology, Faculty of Medicine, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research CentreUniversity of ManitobaWinnipegCanada
  2. 2.Department of Surgery, Section of Cardiac Surgery, St. Boniface General Hospital Cardiac Sciences ProgramUniversity of ManitobaWinnipegCanada
  3. 3.Cardiac StudiesNational Research Council - Institute for BiodiagnosticsWinnipegCanada
  4. 4.Cardiac Sciences ProgramSt. Boniface General HospitalWinnipegCanada

Personalised recommendations