Repeated doses of cardiac mesenchymal cells are therapeutically superior to a single dose in mice with old myocardial infarction

  • Yiru Guo
  • Marcin Wysoczynski
  • Yibing Nong
  • Alex Tomlin
  • Xiaoping Zhu
  • Anna M. Gumpert
  • Marjan Nasr
  • Senthikumar Muthusamy
  • Hong Li
  • Michael Book
  • Abdur Khan
  • Kyung U. Hong
  • Qianhong Li
  • Roberto BolliEmail author
Original Contribution


We have recently demonstrated that repeated administrations of c-kitPOS cardiac progenitor cells (CPCs) have cumulative beneficial effects in rats with old myocardial infarction (MI), resulting in markedly greater improvement in left ventricular (LV) function compared with a single administration. To determine whether this paradigm applies to other species and cell types, mice with a 3-week-old MI received one or three doses of cardiac mesenchymal cells (CMCs), a novel cell type that we have recently described. CMCs or vehicle were infused percutaneously into the LV cavity, 14 days apart. Compared with vehicle-treated mice, the single-dose group exhibited improved LV ejection fraction (EF) after the 1st infusion (consisting of CMCs) but not after the 2nd and 3rd (vehicle). In contrast, in the multiple-dose group, LV EF improved after each CMC infusion, so that at the end of the study, LV EF averaged 35.5 ± 0.7% vs. 32.7 ± 0.6% in the single-dose group (P < 0.05). The multiple-dose group also exhibited less collagen in the non-infarcted region vs. the single-dose group. Engraftment and differentiation of CMCs were negligible in both groups, indicating paracrine effects. These results demonstrate that, in mice with ischemic cardiomyopathy, the beneficial effects of three doses of CMCs are significantly greater than those of one dose, supporting the concept that multiple treatments are necessary to properly evaluate the full therapeutic potential of cell therapy. Thus, the repeated-treatment paradigm is not limited to c-kit POS CPCs or to rats, but applies to other cell types and species. The generalizability of this concept dramatically augments its significance.


Ischemic cardiomyopathy Stem cells Progenitor cells Cell therapy 



This work was supported by NIH Grants P20 GM103492, P01 HL078825 (to RB and MW), and UM1 HL113530 (to RB), and an AHA Scientist Development Grant 13SDG14560005 (to MW).

Supplementary material

395_2017_606_MOESM1_ESM.pptx (521 kb)
Supplementary Fig. 1. CMC cytokine profile. A Proteome Profiler™ Mouse XL Cytokine Array was used to evaluate the secretome of CMCs. Conditioned media from three independent isolations were combined to perform the assay
395_2017_606_MOESM2_ESM.docx (19 kb)
Supplementary material 2 (DOCX 18 kb)


  1. 1.
    Aicher A, Brenner W, Zuhayra M, Badorff C, Massoudi S, Assmus B, Eckey T, Henze E, Zeiher AM, Dimmeler S (2003) Assessment of the tissue distribution of transplanted human endothelial progenitor cells by radioactive labeling. Circulation 107:2134–2139. doi: 10.1161/01.CIR.0000062649.63838.C9 CrossRefPubMedGoogle Scholar
  2. 2.
    Al Kindi A, Ge Y, Shum-Tim D, Chiu RC (2008) Cellular cardiomyoplasty: routes of cell delivery and retention. Front Biosci 13:2421–2434CrossRefPubMedGoogle Scholar
  3. 3.
    Barbash IM, Chouraqui P, Baron J, Feinberg MS, Etzion S, Tessone A, Miller L, Guetta E, Zipori D, Kedes LH, Kloner RA, Leor J (2003) Systemic delivery of bone marrow-derived mesenchymal stem cells to the infarcted myocardium: feasibility, cell migration, and body distribution. Circulation 108:863–868. doi: 10.1161/01.CIR.0000084828.50310.6A CrossRefPubMedGoogle Scholar
  4. 4.
    Blocklet D, Toungouz M, Berkenboom G, Lambermont M, Unger P, Preumont N, Stoupel E, Egrise D, Degaute JP, Goldman M, Goldman S (2006) Myocardial homing of nonmobilized peripheral-blood CD34+ cells after intracoronary injection. Stem Cells 24:333–336. doi: 10.1634/stemcells.2005-0201 CrossRefPubMedGoogle Scholar
  5. 5.
    Bolli R, Patel BS, Jeroudi MO, Li XY, Triana JF, Lai EK, McCay PB (1990) Iron-mediated radical reactions upon reperfusion contribute to myocardial “stunning”. Am J Physiol 259:H1901–H1911PubMedGoogle Scholar
  6. 6.
    Bolli R, Tang XL, Sanganalmath SK, Rimoldi O, Mosna F, Abdel-Latif A, Jneid H, Rota M, Leri A, Kajstura J (2013) Intracoronary delivery of autologous cardiac stem cells improves cardiac function in a porcine model of chronic ischemic cardiomyopathy. Circulation 128:122–131. doi: 10.1161/CIRCULATIONAHA.112.001075 CrossRefPubMedGoogle Scholar
  7. 7.
    Cai C, Guo Y, Teng L, Nong Y, Tan M, Book MJ, Zhu X, Wang XL, Du J, Wu WJ, Xie W, Hong KU, Li Q, Bolli R (2015) Preconditioning human cardiac stem cells with an HO-1 inducer exerts beneficial effects after cell transplantation in the infarcted murine heart. Stem Cells 33:3596–3607. doi: 10.1002/stem.2198 CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Cheng K, Li TS, Malliaras K, Davis DR, Zhang Y, Marban E (2010) Magnetic targeting enhances engraftment and functional benefit of iron-labeled cardiosphere-derived cells in myocardial infarction. Circ Res 106:1570–1581. doi: 10.1161/CIRCRESAHA.109.212589 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Dawn B, Guo Y, Rezazadeh A, Huang Y, Stein AB, Hunt G, Tiwari S, Varma J, Gu Y, Prabhu SD, Kajstura J, Anversa P, Ildstad ST, Bolli R (2006) Postinfarct cytokine therapy regenerates cardiac tissue and improves left ventricular function. Circ Res 98:1098–1105. doi: 10.1161/01.RES.0000218454.76784.66 CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Feldman MD, Erikson JM, Mao Y, Korcarz CE, Lang RM, Freeman GL (2000) Validation of a mouse conductance system to determine LV volume: comparison to echocardiography and crystals. Am J Physiol Heart Circ Physiol 279:H1698–H1707CrossRefPubMedGoogle Scholar
  11. 11.
    Guo Y, Jones WK, Xuan YT, Tang XL, Bao W, Wu WJ, Han H, Laubach VE, Ping P, Yang Z, Qiu Y, Bolli R (1999) The late phase of ischemic preconditioning is abrogated by targeted disruption of the inducible NO synthase gene. Proc Natl Acad Sci USA 96:11507–11512CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Guo Y, Wu WJ, Qiu Y, Tang XL, Yang Z, Bolli R (1998) Demonstration of an early and a late phase of ischemic preconditioning in mice. Am J Physiol 275:H1375–H1387PubMedPubMedCentralGoogle Scholar
  13. 13.
    Hofmann M, Wollert KC, Meyer GP, Menke A, Arseniev L, Hertenstein B, Ganser A, Knapp WH, Drexler H (2005) Monitoring of bone marrow cell homing into the infarcted human myocardium. Circulation 111:2198–2202. doi: 10.1161/01.CIR.0000163546.27639.AA CrossRefPubMedGoogle Scholar
  14. 14.
    Hong KU, Guo Y, Li QH, Cao P, Al-Maqtari T, Vajravelu BN, Du J, Book MJ, Zhu X, Nong Y, Bhatnagar A, Bolli R (2014) c-kit+ Cardiac stem cells alleviate post-myocardial infarction left ventricular dysfunction despite poor engraftment and negligible retention in the recipient heart. PLoS One 9:e96725. doi: 10.1371/journal.pone.0096725 CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Hong KU, Li QH, Guo Y, Patton NS, Moktar A, Bhatnagar A, Bolli R (2013) A highly sensitive and accurate method to quantify absolute numbers of c-kit+ cardiac stem cells following transplantation in mice. Basic Res Cardiol 108:346. doi: 10.1007/s00395-013-0346-0 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Hou D, Youssef EA, Brinton TJ, Zhang P, Rogers P, Price ET, Yeung AC, Johnstone BH, Yock PG, March KL (2005) Radiolabeled cell distribution after intramyocardial, intracoronary, and interstitial retrograde coronary venous delivery: implications for current clinical trials. Circulation 112:I150–I156. doi: 10.1161/CIRCULATIONAHA.104.526749 PubMedGoogle Scholar
  17. 17.
    Keith MC, Bolli R (2015) “String theory” of c-kit(pos) cardiac cells: a new paradigm regarding the nature of these cells that may reconcile apparently discrepant results. Circ Res 116:1216–1230. doi: 10.1161/CIRCRESAHA.116.305557 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Kishore R, Khan M (2016) More than tiny sacks: stem cell exosomes as cell-free modality for cardiac repair. Circ Res 118:330–343. doi: 10.1161/CIRCRESAHA.115.307654 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Li Q, Guo Y, Ou Q, Chen N, Wu WJ, Yuan F, O’Brien E, Wang T, Luo L, Hunt GN, Zhu X, Bolli R (2011) Intracoronary administration of cardiac stem cells in mice: a new, improved technique for cell therapy in murine models. Basic Res Cardiol 106:849–864. doi: 10.1007/s00395-011-0180-1 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Li Q, Guo Y, Ou Q, Cui C, Wu WJ, Tan W, Zhu X, Lanceta LB, Sanganalmath SK, Dawn B, Shinmura K, Rokosh GD, Wang S, Bolli R (2009) Gene transfer of inducible nitric oxide synthase affords cardioprotection by upregulating heme oxygenase-1 via a nuclear factor-{kappa}B-dependent pathway. Circulation 120:1222–1230. doi: 10.1161/CIRCULATIONAHA.108.778688 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Li RC, Ping P, Zhang J, Wead WB, Cao X, Gao J, Zheng Y, Huang S, Han J, Bolli R (2000) PKCepsilon modulates NF-kappaB and AP-1 via mitogen-activated protein kinases in adult rabbit cardiomyocytes. Am J Physiol Heart Circ Physiol 279:H1679–H1689CrossRefPubMedGoogle Scholar
  22. 22.
    Li XY, McCay PB, Zughaib M, Jeroudi MO, Triana JF, Bolli R (1993) Demonstration of free radical generation in the “stunned” myocardium in the conscious dog and identification of major differences between conscious and open-chest dogs. J Clin Invest 92:1025–1041. doi: 10.1172/JCI116608 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Panda NC, Zuckerman ST, Mesubi OO, Rosenbaum DS, Penn MS, Donahue JK, Alsberg E, Laurita KR (2014) Improved conduction and increased cell retention in healed MI using mesenchymal stem cells suspended in alginate hydrogel. J Interv Card Electrophysiol 41:117–127. doi: 10.1007/s10840-014-9940-9 CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Robey TE, Saiget MK, Reinecke H, Murry CE (2008) Systems approaches to preventing transplanted cell death in cardiac repair. J Mol Cell Cardiol 45:567–581. doi: 10.1016/j.yjmcc.2008.03.009 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Sanganalmath SK, Bolli R (2013) Cell therapy for heart failure: a comprehensive overview of experimental and clinical studies, current challenges, and future directions. Circ Res 113:810–834. doi: 10.1161/CIRCRESAHA.113.300219 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Takano H, Bolli R, Black RG Jr, Kodani E, Tang XL, Yang Z, Bhattacharya S, Auchampach JA (2001) A(1) or A(3) adenosine receptors induce late preconditioning against infarction in conscious rabbits by different mechanisms. Circ Res 88:520–528CrossRefPubMedGoogle Scholar
  27. 27.
    Tang XL, Li Q, Rokosh G, Sanganalmath S, Chen N, Ou Q, Stowers H, Hunt G, Bolli R (2016) Long-term outcome of administration of c-kitPOS cardiac progenitor cells after acute myocardial infarction: transplanted cells do not become cardiomyocytes, but structural and functional improvement and proliferation of endogenous cells persist for at least one year. Circ Res 118:1091–1105. doi: 10.1161/CIRCRESAHA.115.307647 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Tang XL, Li Q, Rokosh G, Sanganalmath SK, Chen N, Ou Q, Stowers H, Hunt G, Bolli R (2016) Long-term outcome of administration of c-kit(POS) cardiac progenitor cells after acute myocardial infarction: transplanted cells do not become cardiomyocytes, but structural and functional improvement and proliferation of endogenous cells persist for at least one year. Circ Res 118:1091–1105. doi: 10.1161/CIRCRESAHA.115.307647 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Tang XL, Qiu Y, Park SW, Sun JZ, Kalya A, Bolli R (1996) Time course of late preconditioning against myocardial stunning in conscious pigs. Circ Res 79:424–434CrossRefPubMedGoogle Scholar
  30. 30.
    Tang XL, Rokosh G, Sanganalmath SK, Tokita Y, Keith MC, Shirk G, Stowers H, Hunt GN, Wu W, Dawn B, Bolli R (2015) Effects of intracoronary infusion of escalating doses of cardiac stem cells in rats with acute myocardial infarction. Circ Heart Fail 8:757–765. doi: 10.1161/CIRCHEARTFAILURE.115.002210 CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Tang XL, Rokosh G, Sanganalmath SK, Yuan F, Sato H, Mu J, Dai S, Li C, Chen N, Peng Y, Dawn B, Hunt G, Leri A, Kajstura J, Tiwari S, Shirk G, Anversa P, Bolli R (2010) Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction. Circulation 121:293–305. doi: 10.1161/CIRCULATIONAHA.109.871905 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Terrovitis J, Lautamaki R, Bonios M, Fox J, Engles JM, Yu J, Leppo MK, Pomper MG, Wahl RL, Seidel J, Tsui BM, Bengel FM, Abraham MR, Marban E (2009) Noninvasive quantification and optimization of acute cell retention by in vivo positron emission tomography after intramyocardial cardiac-derived stem cell delivery. J Am Coll Cardiol 54:1619–1626. doi: 10.1016/j.jacc.2009.04.097 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Tokita Y, Tang XL, Li Q, Wysoczynski M, Hong KU, Nakamura S, Wu WJ, Xie W, Li D, Hunt G, Ou Q, Stowers H, Bolli R (2016) Repeated administrations of cardiac progenitor cells are markedly more effective than a single administration: a new paradigm in cell therapy. Circ Res 119:635–651. doi: 10.1161/CIRCRESAHA.116.308937 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Wysoczynski M, Dassanayaka S, Zafir A, Ghafghazi S, Long BW, Noble C, DeMartino AM, Brittian KR, Bolli R, Jones SP (2016) A new method to stabilize c-kit expression in reparative cardiac mesenchymal cells. Front Cell Dev Biol 4:78. doi: 10.3389/fcell.2016.00078 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Wysoczynski M, Guo Y, Moore J, Muthusamy M, Li Q, Nasr M, Li H, Nong Y, Wu W, Tomlin A, Zhu X, Hunt G, Gumpert A, Book M, Khan A, Tang XL, Bolli R (2016) A new population of cardiac mesenchymal cells isolated on the basis of adherence: phenotype and reparative properties. J Am Coll Cardiol (2016) (in press)Google Scholar
  36. 36.
    Yu J, Du KT, Fang Q, Gu Y, Mihardja SS, Sievers RE, Wu JC, Lee RJ (2010) The use of human mesenchymal stem cells encapsulated in RGD modified alginate microspheres in the repair of myocardial infarction in the rat. Biomaterials 31:7012–7020. doi: 10.1016/j.biomaterials.2010.05.078 CrossRefPubMedGoogle Scholar
  37. 37.
    Zhu WX, Myers ML, Hartley CJ, Roberts R, Bolli R (1986) Validation of a single crystal for measurement of transmural and epicardial thickening. Am J Physiol 251:H1045–H1055PubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Yiru Guo
    • 1
  • Marcin Wysoczynski
    • 1
  • Yibing Nong
    • 1
  • Alex Tomlin
    • 1
  • Xiaoping Zhu
    • 1
  • Anna M. Gumpert
    • 1
  • Marjan Nasr
    • 1
  • Senthikumar Muthusamy
    • 1
  • Hong Li
    • 1
  • Michael Book
    • 1
  • Abdur Khan
    • 1
  • Kyung U. Hong
    • 1
  • Qianhong Li
    • 1
  • Roberto Bolli
    • 1
    Email author
  1. 1.Institute of Molecular CardiologyUniversity of Louisville School of MedicineLouisvilleUSA

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