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INNOVATIVE METHODS OF APPLICATION OF STEM CELLS IN MEDICINE

POLISH PROJECT ON VERY SMALL EMBRYONIC-LIKE STEM CELLS (VSELs) IN CARDIOLOGY. INNOVATIVE METHODS OF APPLICATION OF STEM CELLS IN MEDICINE. Michał Tendera MD Wojciech Wojakowski MD THIRD DIVISION OF CARDIOLOGY MEDICAL UNIVERSITY OF SILESIA, KATOWICE, POLAND. VSELs.

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INNOVATIVE METHODS OF APPLICATION OF STEM CELLS IN MEDICINE

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  1. POLISH PROJECT ON VERY SMALL EMBRYONIC-LIKE STEM CELLS (VSELs) IN CARDIOLOGY • INNOVATIVE METHODS OF APPLICATION OF STEM CELLS IN MEDICINE Michał Tendera MD Wojciech Wojakowski MD THIRD DIVISION OF CARDIOLOGY MEDICAL UNIVERSITY OF SILESIA, KATOWICE, POLAND

  2. VSELs Cytometry A. 2009 January; 75(1): 4–13.

  3. IMAGE STREAM Cytometry A. 2009 January; 75(1): 4–13.

  4. 2mm 1mm 2mm 4mm 1mm 1mm 2mm 2.5mm TEM of murine Sca-1+lin- CD45- (VSEL) and Sca-1+lin- CD45+ (HSC) cells Sca-1+lin-CD45- A B Sca-1+lin-CD45+ Kucia et al. Leukemia 2006,20:857-869

  5. REGULATION OF QUIESCENCE AND PLURIPOTNCY VSELs ARE PROGENY OF EPIBLAST-DERIVED CELLS Shinet al. Leukemia, 2009:23(11): 2042

  6. REGULATION OF QUIESCENCE AND PLURIPOTNCY RECOVERY OF GENOMIC IMPRINTING DURING FORATION OF VSEL-DS Shinet al. Leukemia, 2009:23(11): 2042

  7. VSEL-DERIVED CM GFP+ VSEL MURINE VSEL-DERIVED CM C2C12 myoblasts 7 days 5x104 CELLS DMEM + 2% FBS 37oC, 5% CO2. C2C12 CARDIAC DIFFERENTIATION Tripsinization Re-sorting C2C12 GFP+ VSEL Wojakowski et al. Int J Onc, 2010, inpress

  8. 120 MNC VSEL DAY 4 100 DAY 6 DAY 9 DAY 12 80 DAY 16 (fold-difference) 60 mRNA 40 20 0 Nkx 2.5/Csx GATA-4 Troponin I Mybpc3 actinin 2 actinin 3 VSEL-DERIVED CM Wojakowski et al. Int J Onc, 2010, inpress

  9. SORT I BM isolation VSEL Expansion culture EGFP transgenic mice [C57BL/6] VSELs 9 days C2C12 feeder layer HCSs SORT II WT mice [C57BL/6] after infarction Cardiac pre-differen. Injection 6 months of follow-up 5 days Injection MURINE MODEL OF ACUTE MI

  10. † † † † † * * * * * BSL BSL BSL 48 h 48 h 48 h 1 mo 1 mo 1 mo 3 mo 3 mo 3 mo 6 mo 6 mo 6 mo Gr I (Vehicle) Gr II (HSCs) Gr III (VSELs expanded and pre-differentiated) IMPROVEMENT OF LV FUNCTION LV Ejection Fraction LV End-systolic Diameter *P < 0.05 vs. Vehicle-treated †P < 0.05 vs. HSCs † % mm Infarct Wall Thickness (Diastole) mm Zuba-Surma EK et al. Circulation (AHA 2008)

  11. EGFP+ MYOCYTES AFTER 6 MONTHS Zuba-Surma EK et al. Circulation (AHA 2008)

  12. VSEL-DERIVED CM STUDY LIMITATIONS • limited gene-array and no proteomic data • no EP data • murinetransplantation model RESEARCH PLAN • comparisionwithESC-derived CM throughoutdifferentiation • electrophysiologystudy • differentiation of humanBM-VSELs • implantation of EGFP+ VSELsinmurine model of reperfused MI

  13. HUMAN VSELS Cytometry A. 2009 January; 75(1): 4–13.

  14. CIRCULATION OF VSELs Peripheral Blood Bone Marrow Skeletal Muscle Other Tissues MYOCARDIAL INFARCTION MOBILIZATION OF VSELS Homing/Engraftment Mobilization MOBILIZATION OF CXCR4+ CELLS Increased Expression of Cardiac/Endothelial Markers Kucia et al.. Circulation Res. 2004, 95:1191-9 Ratajczak et al.. Biol. Cell 2005, 97:113-146. Wojakowski et al. Heart 2008 94(1):27-33 Wojakowski, W. et al. J Am Coll Cardiol 2009;53:1-9

  15. Monocytes 12.4µm Granulocytes 13.5µm Lymphocytes 8.4µm Erythrocytes 7.5µm VSEL 7.04 µm HUMAN VSELs Wojakowski, W. et al. J Am Coll Cardiol 2009;53:1-9

  16. MOBILIZATION OF VSELs MOBILIZATION OF VSELs INDUCED BY ISCHEMIC STROKE Paczkowska, E. et al. Stroke 2009;40:1237-1244

  17. MOBILIZATION BY PHYSICAL EXERCISE HEALTHY SUBJECTS AND PATIENTS WITH FIRST ACUTE MI <45y • HYPOTHESIS : MOBILIZATION OF VSELs AND THEIR FUNCTION IS REDUCED IN PATIENTS WITH HEART FAILURE

  18. MOBILIZATION MOBILIZATION OF VSELS DURING HEART SURGERY AIMS • MOBILIZATION OF VSELs DURING • CARDIAC SURGERY • CHILDREN VS. ADULTS • GENE EXPRESSION PROFILE • ON-PUMP VS. OFF-PUMP • PRESENCE OF VSELs IN HUMAN • HEART SDF-1 Mieno, S. et al. Circulation 2006;114:186-192

  19. Images of Oct-4+ VSELs from organs – by ImageStream system Oct-4 and 7-AAD Bone marrow 10m Thymus Spleen Oct-4 and 7-AAD Heart Brain Lungs Pancreas Testes Kidneys Liver Skeletal muscles

  20. PT SELECTION RANDOMIZATION UNSELECTED SELECTED CONTROL CLINICAL APPLICATION LABELLING ANTI-CD34 REGENT I STEP MRI LABELLING ANTI-CXCR4 VENTRICULO VENTRICULO II STEP 6 MONTHS CELL INFUSION CELL INFUSION CD34+CXCR4+ Tendera et al..Eur Heart J. 2009 Jun;30(11):1313-21. MRI + VENTRICULO + CLINICAL F-U MRI + CLIN F-U

  21. CLINICAL APPLICATION REGENT 34.5 36 -4 3 5 31 29.5 Tendera et al..Eur Heart J. 2009 Jun;30(11):1313-21. Wilcoxon test

  22. CLINICAL APPLICATION INTRACORONARY RADIOLABELLED SELECTED 34+ CELLS 99mTc-extametazime-labeled Musiałek et al., TCT 2009

  23. REGENT-VSELs „Randomized, prospective, double-blinded, placebo controlledtrial to assesstheeffects of BM-derivedimmunoselected CD133+ cells on LV perfusion and function inpatientswithinducible ischemia and refractory angina” AIMS • Efficiency of intracoronaryinfusion of BM-derived CD133+ cellsaftersuccesfulrecanalization of the CTO of thearteryinpatientswithreractory angina and incucible ischemia. • Efficiency of NOGA-guideddirectintramyocardialinjection of BM-derived CD133+ cellsinpatientswithreractory angina and incucible ischemia ineligible for recanalization of CTO.

  24. PODZADANIE 7.5 INCLUSION CRITERIA 60 patientswith CTO inqualifyingangiogramscheduled for PCI Inclusioncriteria: angina CCS class ≥2, symptomsdespiteoptimalmedicaltherapy, chronictotalocclusion of thearterysupplyingviablemyocardium, LVEF ≤ 50%, ineligible for surgicalrevascularization Exclusioncriteria: acutecoronarysyndrome, myocardialinfarction< 3 months, LVEF <30%, renalfailure (GFR <30 mL/min/1.73m2), malignancy, bleedingdiathesis

  25. PROTOCOL SCREENING INFORMED CONSENT • Clinicalevaluation: CCS, NYHA, quality of life • Exercise test • SPECT imaging • MRI BASELINE CTO PCI SUCCESSFUL UNSUCCESSFUL CELLS ISOLATION (CliniMACS) RANDOMIZATION INTRACORONARY CD133+ CELLS PLACEBO INTRAMYOCARDIAL CD133+ CELLS PLACEBO • Clinicalevaluation: CCS, NYHA, quality of life • Exercise test • SPECT imaging • MRI 3 MONTHS

  26. PODZADANIE 7.5 PROGRESSION OF ATHEROSCLEROSIS INRAVASCULAR ULTRASOUND WITH VH OPTICAL COHERENCE TOMPGRAPHY MICROCIRCULATORY FUNCTION ENDOTHELIAL REACTIVITY 30 Warszawa, 09.01.2010 Innowacyjne metody wykorzystania komórek macierzystych w medycynie

  27. SUMMARY • CARDIAC DIFFERENTIATION OF BM-DERIVED VSELS • MOBILIZATION AND FUNCTION OF CIRCULATING VSELS IN PATIENTS WITH HF, MI <45y AND UNDERGOING CARDIAC SURGERY • PHYSICAL EXERCISE AND MOBILIZATION • PRESENCE OF VSELs IN HUMAN HEARTS AND ARTERIES • CLINICAL TRIAL

  28. TEAM III DIVISION OF CARDIOLOGY MEDICAL UNIVERSITY OF SILESIA BONE MARROW TRANSPLANT UNIT DEPARTMENT OF HEMATOLOGY MEDICAL UNIVERSITY OF SILESIA DEPARTMENT OF CARDIAC SURGERY MEDICAL UNIVERSITY OF SILESIA DEPARTMENT OF CHILDREN CARDIAC SURGERY JAGIELLONIAN UNIVERSITY STEM CELL INSTITUTE UNIVERSITY OF LOUISVILLE DEPARTMENT OF BIOTECHNIOLOGY JAGIELLONIAN UNIVERSITY

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