Stem Cell Therapy for Heart Disease Revisited: the Paracrine Paradigm

Transcription

1 17 th ISCT Annual Meeting May 18-21, Rotterdam, The Netherlands Stem Cell Therapy for Heart Disease Revisited: the Paracrine Paradigm Massimiliano Gnecchi, MD, PhD Università degli studi di Pavia Fondazione IRCCS Policlinico San Matteo

2 Cell therapy for the heart Gnecchi M - ISCT Gnecchi 211 M et al. in Heart Development and Regeneration Edited by Rosenthal and Harvey Elsevier

3 Mesenchymal stem cells Gnecchi M et al. in Heart Development and Regeneration Edited by Rosenthal and Harvey Elsevier

4 Possible mechanisms of MSC action Mesenchymal stem cell Differentiation or cell fusion Differentiation or cell fusion Cardiomyocyte VSMC Endothelial cell Vasculogenesis Cardiomyocyte regeneration Vascular regeneration

5 Possible mechanisms of MSC action Mesenchymal stem cell Differentiation or cell fusion Differentiation or cell fusion Cardiomyocyte VSMC Endothelial cell Vasculogenesis Cardiomyocyte regeneration Vascular regeneration

6 β-gal Merge β-mhc α-actinin Desmin ctnt β-gal α-actinin Phospholamban Toma C et al, Circulation 22; 15:93-98

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8 Cell Differentiation Cell fusion 3 days 3 days 7 days 14 days Noiseux N et al, Mol Ther 26; 14:84-85

9 Possible mechanisms of MSC action Mesenchymal stem cell Differentiation or cell fusion Differentiation Or cell fusion Cardiomyocyte VSMC Endothelial cell Vasculogenesis Cardiomyocyte regeneration Vascular regeneration

10 Circulation 25; 111: MSCs differentiate into endothelial cells MSCs differentiate into VSMC Factor VIII Dil MSC Dil MSC Merge nuclei α-smooth muscle actin

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12 Modified form Loffredo F. Circ Res 28;13:128

13 Possible mechanisms of MSC action Mesenchymal stem cell Differentiation or cell fusion Differentiation or cell fusion Cardiomyocyte Low efficiency VSMC Endothelial cell Vasculogenesis Cardiomyocyte regeneration Vascular regeneration

14 Possible mechanisms of MSC action Mesenchymal stem cell Differentiation or cell fusion Differentiation or cell fusion Cardiomyocyte VSMC Endothelial cell Vasculogenesis Cardiomyocyte regeneration PARACRINE HYPOTHESIS Vascular regeneration

15 Paracrine hypothesis Adult Stem Cell Autocrine effects Release of soluble factors PARACRINE EFFECTS Myocardial protection Cardiac regeneration Cardiac metabolism Contractility Cardiomyocytes Endothelial cells Smooth muscle cells Fibroblasts Cardiac stem cells Neovascularization Cardiac remodeling Modified from: Gnecchi M et al. Circ Res 28

16 Paracrine hypothesis Adult Stem Cell Autocrine effects Release of soluble factors PARACRINE EFFECTS Myocardial protection Cardiac regeneration Cardiac metabolism Contractility Cardiomyocytes Endothelial cells Smooth muscle cells Fibroblasts Cardiac stem cells Neovascularization Cardiac remodeling Modified from: Gnecchi M et al. Circ Res 28

17 Adjacent Remote dutp-labeled Myocyte Nuclei/ h 1-2d 7d 14d 1m h 1-2d 7d 14d 1m Cheng W et al, Exp Cell Res 1996; 226:

18 Testing conditioned medium Gnecchi M and Melo LG in Stem Cells in Regenerative Medicine - Humana Press

19 Cytoprotective effects of CM in vitro 6 Caspase 3 Activity Baseline Control M Relative Units 3 CTR-M GFP H-M Akt H-M Number of ARCVs/field GFP H-M Akt H-M Total ARVMs Rod-shaped ARVMs Baseline CTR-M GFP H-M Akt H-M TUNEL-(+) ARVCs ARVC nuclei CTR-M TUNEL assay GFP H-M Akt H-M Gnecchi M et al, Nature Medicine 25

20 CM effects on ischemic myocardium Infarct size (% LV) TTC CTR-CM GFP-CM Lectin I Gnecchi M et al, FASEB J 26 Akt-CM Capillary vessels/field Apoptotic index TUNEL staining CTR-CM GFP-CM Akt-CM Capillary density CTR-CM GFP-CM Akt-CM

21 Modified from Circ Res 26; 98: Normoxia Hypoxia VEGF Pg/µg protein Apoptosis index (%) 16 8 DMEM MSC MSC pre exposed to hypoxia bfgf IGF SDF Pg/µg protein Pg/µg protein Pg/µg protein BM-M CM-M BM-M CM-M

22 Paracrine hypothesis Adult Stem Cell Autocrine effects Release of soluble factors PARACRINE EFFECTS Myocardial protection Cardiac regeneration Cardiac metabolism Contractility Cardiomyocytes Endothelial cells Smooth muscle cells Fibroblasts Cardiac stem cells Neovascularization Cardiac remodeling Modified from: Gnecchi M et al. Circ Res 28

23 Endogenous cardiac regeneration Transplanted hbmscs enhance myogenesis by augmenting host CMC proliferation and differentiation Ki67 index (%) hbmsc TBMC PBS Modified from: Yoon Y et al, JCI 25; 115:

24 MSC stimulates endogenous Cardiac Stem Cells Hatzistergos KE et al, Circ Res 21; 17:

25 MSC stimulates endogenous CMCs cell cycling Hatzistergos KE et al, Circ Res 21; 17:

26 Gnecchi M et al. in Heart Development and Regeneration Edited by Rosenthal and Harvey Elsevier - 21

27 What is next? Which are the mediators? Are paracrine effects cell specific? How do we take paracrine to the bedside? Do we still need stem cells to repair the heart?

28 Which are the mediators? Production and release of SF Transcriptomic

29 GFP Akt Approximately 65 transcripts were differentially regulated Focus on: Cytoprotective genes Among the 65 transcripts we identified 51 unique genes encoding for known secreted factors that might potentially account for the cytoprotective effects observed

30 Sfrp2 is upregulated in Akt-MSCs Secreted frizzeled related proteins (Sfrp) are Wnt antagonists that share a similar CRD with Fz receptor Gene expression Protein expression 1 GFP-MSC Akt-MSC Normoxia mrna Expression 5 Sfrp2 Sfrp2 Sfrp1 Sfrp3 1 Akt-MSC 2 GFP-MSC 3 Akt-MSC + LY GFP-MSC + LY2942 Mirotsou M et al. PNAS 27; 14:

31 In vitro effects of Sfrp2 In vivo effects of Sfrp2 2.5 Caspase activity (arbitrary units) Caspase activity (arbitrary units) pm Sfrp2 Normoxia Normoxia # Ctr CM Hypoxia Akt CM 24h Hypoxia Akt CM (-) Sfrp2 Infarct size (% LV) PBS Akt-CM Akt-CM (-)Sfrp2 # Mirotsou M et al. PNAS 27; 14:

32 PNAS 28

33 April 29 Sfrp2 and myocardial infarction

34 Which are the mediators? Transcriptomic Production and release of SF Proteomic

35 LC-MS/MS (low sensitivity for cytokines) 247 proteins encoded by 132 unique known genes. 29 unknown proteins

36 Cytokine arrays 72 cytokines/growth factors were found to be reproducibly secreted by MSCs

37 Biological processes modulated by MSC-secreted proteins

38 What is next? Which are the mediators? Combined Are paracrine transcriptomic effects cell specific? and proteomic approach identifies putative paracrine How do we take paracrine to the bedside? factors of fetal mesenchymal stem cells Dott.ssa we Danieli still need P. stem cells to repair the heart? Saturday 5/21/211 Time:

39 What if pracrine mediators were not proteins or pepties? Transcriptomic Production and release of SF Proteomic Transcriptomic & Proteomic Exosomes Sequencing, mirna Array & Real-Time PCR Delivery of SF and mirna

40 In vivo effects of CM subfractions Error Bars: +/- 1 SE n=6/group The MSC-CM contains exosomes 4 3 Saline HEK293 CM <1 KDa IS/AAR (%) 2 1 <3 KDa <5 KDa <1 KDa >1 KDa Lai et al. Stem Cell Research 21; 4:

41 Isolation of exosome subfraction In vivo effects of purified MSC-derived exosomes 4 Error Bars: +/- 1 SE IS/AAR (%) Saline (n=1) CM (n=6) HPLC-F1 (n=5) 5 Error Bars: +/- 1 SE 4 AAR/LV (%) Saline (n=1) CM (n=6) HPLC-F1 (n=5) Lai et al. Stem Cell Research 21; 4:

42 What is next? Which are the mediators? Are paracrine effects cell specific? How do we take paracrine to the bedside? Do we still need stem cells to repair the heart?

43 Cardiac Progenitors Type of progenitor Reference c-kit + cardiac progenitor cells Beltrami et al, Cell 23 Bearzi et al, PNAS 27 Bearzi et al, PNAS 29 Side Population myocardial cells Oh et al, PNAS 23 Progenitor cells Sca-1 + Matsuura et al, J Biol Chem 24 Smits et al, Nat Protoc 29 Cardiosphere-derived Cells Messina et al, Circ Res 24 Smith et al, Circulation 27 Cardioblast Isl1 + Laugwitz et al, Nature 25 Moretti et al, Cell 26 Epicardial tissue stem cells Limana et al, Circ Res 27

44 Relative roles of direct regeneration versus paracrine effects of human Cardiosphere-Derived Cells transplanted into infarcted mice Chimenti et al, Circ Res 21 Pro-survival in vitro effects of CDC-CM on NRVMs exposed to hypoxia Pro-angiogenic in vitro effects of CDC-CM on HUVECs % of early apoptotic cells CDC-CM 24h NhDF-CM CTRL-M 72h CDC-CM NhDF-CM CTRL-M EGM Matrigel Assay CTRL-M CDC-CM NHDF-CM % of CDC-CM effect p<.5 ; p<.1 vs. CDC CDC-CM CDC-CM Anti-VEGF CDC-CM Anti-HGF CDC-CM both NhDF-CM [pixels] EGM CDC-CM NhDF-CM CTRL-M p<.5 ; p<.1 vs. CDC % of CDC-CM effect CDC-CM CDC-CM Anti-VEGF CDC-CM Anti-HGF CDC-CM both NhDF-CM

45 Paracrine works also in vivo Human CDC trasplantation into infarcted mice Chimenti et al, Circ Res 21 Cardioprotection Angiogenesis Paracrine contribution TUNEL + /mm # Capillaries/nuclei Capillaries/nuclei MHC + /mm # Human No Human CDC NhDF PBS CDC NhDF PBS CDC NhDF CDC NhDF p<.1; # p<.5 vs. control group p<.1; # p<.5 vs. CDC

46 Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 3-day old infarction Tang et al, Circulation 21 Exogenous c-kit+ CSC administration induce endogenous CSC proliferation 4 c-kit + /Nkx + /MHC - cells 8 c-kit + /Nkx + /MHC + cells Vehicle EGFP neg EGFP pos Cells/1. nuclei Risk Noninfarcted Risk Noninfarcted

47 c-kit + GATA-4 high rat cardiac stem cells foster adult cardiomyocyte survival through IGF-1 paracrine signalling Kawaguchi et al, Plos ONE 21 c-kit + GATA-4 mediate cytoprotection Apoptotic Myocyte Nuclei (%) TUNEL assay days Apoptotic Myocyte (%) Caspase3 activation CMC alone Fibroblast + CMC CSC GATA-4 - CSC GATA-4 + CMC alone Fibroblast + CMC CSC GATA-4 - CSC GATA-4 + p<.5 vs. CMC alone, Fibroblast + CMC and CSC GATA-4 -

48 Cardiac Progenitors Autocrine effects CSC ckit + Release of soluble factors PARACRINE EFFECTS Myocardial protection CDC CSC ckit + GATA4 + Cardiac metabolism Contractility CDC CSC ckit + GATA4 + Cardiomyocytes Endothelial cells Smooth muscle cells Fibroblasts Cardiac stem cells Cardiac regeneration CDC CSC ckit + Neovascularization CDC CSC ckit + Cardiac remodeling CSC ckit +

49 What is next? Which are the mediators? Are paracrine effects cell specific? How do we take paracrine to the bedside? Do we still need stem cells to repair the heart? The answer at the 18 th ISCT meeting in 212

50 Acknowledgements Laboratory of Experimental Cardiology for Cell and Molecular Therapy MY AMERICAN FRIENDS Victor J Dzau Luis G Melo Fondazione IRCCS San Matteo, Pavia Joanne S Ingwall Richard E Pratt Abeel Mangi Elisabetta Cervio Patrizia Danieli Manuela Mura Francesco Copes Chiara Ciuffreda Federica Pisano Giuseppe Malpasso Huamei He Lunan Zhang Nicholas Noiseux Hui Mu Olin Liang Fulvio Morello Maria Mirotsou Arjun Deb COLLABORATORS Peter J Schwartz Antonio Beltrami (Udine) Daniela Cesselli (Udine) Laura Calvillo Paolo Bernasconi Marina Boni Gianluca Viarengo Cesare Perotti Claudia Del Fante Theo Luider (Erasmus University, Rotterdam, NL) Rudolf De Boer (Groningen University, NL) Tiziano Moccetti (Lugano) Angelo Auricchio (Lugano) Lucia Turchetto (Lugano) Marina Radrizzani (Lugano) Vittorio Rosti Marianna Roccio Contact: m.gnecchi@unipv.it