在体干细胞生物显像和心肌修复

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1、In Vivo Cellular and Molecular MRI of Embryonic Stem Cell Survival and Proliferation Phillip C. Yang, MD Assistant Professor Division of Cardiovascular Medicine Stanford University (No disclosure or conflict of interest) GREAT WALL INTERNATIONAL CONGRESS OF CARDIOLOGY BEIJING, CHINA 2008 I. Stem Cel

2、l Therapy: Congestive Heart Failure Coronary artery disease: #1 killer in the United States 1.1 million new or recurrent MI and 500,000 deaths per year Improved acute MI therapy shift in the landscape Congestive heart failure: #1 cause of hospital admissions in the US Prevalence: 5 million patients

3、Incidence: 300,000 patients/year Advanced therapy: 5-year survival 50% Heart transplant: 1,000 pt/year Recent stem cell research: alternative therapeutic option? II. Evaluation of Cell Therapy Clinical trials: modest, temporary improvement in LVEF Cell survival following transplantation into the hea

4、rt Fundamental biology of cell therapy: Restore & regenerate In vivo imaging of stem cell viability III. In Vivo Multi-Modality Imaging MRI RN BLI Sensitivity 10-4 - 10-9 Mole/L 10-11 -10-12 Mole/L 10-15 - 10-17 Mole/L Spatial resolution 500 m 3-5 mm 3-5mm Temporal resolution 10 ms seconds seconds (

5、Hendry et al, JCTS, 2008) In vivo molecular and cellular MRI Cell Nucleus Vector (Delivery Vehicle) Protein Reporter Gene Iron-oxide labeld MAb IRES CMV/ubiquitin IgK HA/myc PDGFR-TM GFP luc mRNA PLL PS PS PS PLL PS PLL PLL Iron-oxide HA myc Manganese Ca2+-channel IV. Cellular MRI: iron-oxide H&E Pr

6、us blue H&E 40 x P blue 40 x (Hung et al, Circulation: Cardiovascular Img. 2008) (Suzuki et al, MRM, 2007) Metabolic effects: 3 transfection methods In vivo MRI of myocardial restoration End-diastole End-systole MI control Non-treated heart Treated heart (Hendry et al, JCTS 2008) mESC mESC Fibroblas

7、t EF Comparison at week400.10.20.30.40.50.6(a)(b)(c)(d)(e)Cellular survival (Cunningham et al, Magn Reson Med 2005) Survival d2 d5 d15 d2 d5 d7 GRE OR In vivo serial survival and proliferation 1POD 4POD 7POD 10POD 16POD 4 5 6 (Suzuki et al, MRM, accepted, 2008) Control 0.01 0.05 0.10 0.50 1.00 (mM)

8、p/sec/cm2/sr V. Cellular MRI: Manganese 3.00 0.01 with Dead cells (Yamada et al, MRM, submitted) Biological modulation of Ca2+-channel activity control Mn Mn + BK Mn+Vera 00.20.40.60.811.21.40.1 mMVeraBK727 379 1220 97.2 147.6 Non-contractile embryoid body Contractile embryoid body In vivo Mn2+ MRI

9、of myocardial survival and teratoma VI. In vivo molecular imaging: Reporter Gene Cell Nucleus DNA Reporter Gene Construct Promoter Reporter Gene Vector (Delivery Vehicle) mRNA Reporter Protein Probe Detector Signal In vivo molecular MRI: Survival USPIO-monoclonal Ab against HA & c-myc MRI reporter g

10、ene driven by CMV/ubiquitin promoter Cloning into p2k7-RG lentivirus Ubc EF1 p2k7(neo or blas) Ubc EF1 (Chung et al, Nat Biotech, 2008, in prep) Validation: FACS c-myc expression HA expression mESC hESC Validation: Immunohistology Anti c-myc FITC DAPI Prussian blue Validation: In vitro BLI and MRI m

11、ESC mESC + anti c-myc mESC-RG + anti c-myc mESC-RG + anti HA mESC + anti HA mESC-RG hESC + anti c-myc hESC-RG + anti c-myc hESC + anti HA hESC-RG + anti HA In vivo MRI of hESC-RG VII. Conclusion In vivo cellular and molecular MRI: iron-oxide, manganese, and reporter gene Robust labeling Safety profi

12、le Dual imaging High sensitivity: longitudinal tracking of location, survival, and teratoma High spatial and temporal resolution: myocardial restoration Stanford Laboratory for Stanford Laboratory for Cardiovascular RegenerationCardiovascular Regeneration Yang Laboratory Takayasu Arai Jaehoo Chung R

13、ajesh Dash Pratima Kundu Yoriyasu Suzuki Mayumi Yamada Hunter Wang CT Surgery Stephen Hendry Grant Hoyt Ahmed Sheikh Robert Robbins CV Molecular Biology Thomas Quertermous CV Imaging Feng Cao Chuck Cunningham Tim Doyle Sam Gambhir Michael McConnell John Pauly Masahiro Terashima Joe Wu Esther Xie Pathology Andrew Connolly CV Intervention Todd Brinton Peter Fitzgerald Alan Yeung Paul Yock Stem Cell Biology Renee Reijo-Pera Micha Drukker Irv Weissman Funding Source NIH AHA CIRM