Human Embryonic Stem Cells for Cardiac Regeneration
Transcription
Human Embryonic Stem Cells for Cardiac Regeneration
Human Embryonic Stem Cells for Cardiac Regeneration Robert A. Kloner, MD, PhD Professor of Medicine Keck School of Medicine at University of Southern California Director of Research Heart Institute, Good Samaritan Hospital Los Angeles, CA TCT; Sep 2010 Washington, DC Conflict of Interest Dr. Kloner’s lab receives a grant form Celavie for Stem Cell Research Several recent exciting findings suggest that: Cardiomyocytes derived from human embryonic stem cells may form stable grafts in myocardial ischemia/infarction models and contribute to improved LV function Cardiac progenitor cells can be isolated and transplanted into myocardial infarction models improving LV function Humoral factors (hepatocyte growth factor, insulin-like growth factor) may stimulate local cardiac progenitor cells to improve LV function Pluripotent stem cells induced from skin fibroblasts could generate functional cardiac myocytes (would negate the need for immunosupression) BUT … there are hurdles to overcome: ─ small graft size in some studies ─ cell death-necrosis vs. apoptosis or both ─ poor retention of cells ─ rejection ─ true contribution to contraction ─ only transient improvement in LV function Methods In recipient athymic nude rats, the proximal left anterior descending coronary artery was occluded for 15 minutes; then the coronary artery was reperfused (ischemic with minimal infarction). A cell suspension of two million cells containing GFP positive beating immature cardiomyocytes derived from human embryonic stem cells was injected directly into the myocardium within the ischemic risk area at 5 minutes after left coronary artery occlusion. The hearts were harvested at various time points (hours, 1, 2 or 4 weeks) later and processed for histology, immunohistochemical staining, and fluorescence and immunofluorescence microscopy. Results In the nude rats, transplanted hESC-derived cardiomyocytes formed grafts in 9 out of 10 hearts demonstrated by H&E staining, immunohistochemical staining and GFP epifluorescence. Cells that demonstrated GFP epi-fluorescence also stained positive (co-localized) for the muscle marker alpha-actinin and exhibited cross striations (sarcomeres). Furthermore cells that stained positive for the antibody to GFP (immuno-histochemistry) also stained positive for the muscle marker sarcomeric actin. A E B C F D G Transplanted hESC-CMs form stable grafts in vivo in nude rat (4 weeks post-transplantation) A. Low power image of GFP epi-fluorescence (green) in a heart engrafted with hESC-derived cardiomyocytes. B-D. GFP epi-fluorescence (green; panel B, alpha-actinin immune reactivity (red, rhodamine-conjugated secondary antibody; panel C) and a merged image showing both signals (panel D) Transplanted hESC-CMs form stable grafts in vivo in nude rat (4 weeks posttransplantation). Green cells represent the transplant. Note their sarcomeres and smaller diameter compared to native host myocardial cells on the right. Results At 4 weeks after transplantation, engrafted hESC-derived cardiomyocytes expressed connexin 43, suggesting the presence of nascent gap junctions between donor and host cells. A B C D E F At 4 weeks engrafted hESC-CMs expressed connexin 43 in nude rat Results No significant inflammatory response was observed in the myocardium of nude rats that received hESC-derive cardiomyocytes at 4 weeks after transplantation. In contrast, hESC-derived cardiomyocytes injected into immune competent SpragueDawley rats resulted in an overt lymphocytic infiltrate typical of rejection. A C B D hESC-derived cardiomyocytes injected into immune competent Sprague-Dawley rats resulted in an overt lymphocytic infiltrate typical of rejection at 4 weeks after transplantation. Improvements in LVEF in Infarct Models Receiving h-ESCs Author Reference Model LV Function Results Kofidis et al. European J. Cardiothoracic Surgery 2005 Mouse MI Model MRI-3 weeks EF-control 44% EF cells 58% (p = ?) Author Reference Model LV Function Results Laflamme et al. Nature Biotech 2007 Rat MI model MRI - 4 week EF-control 42-45% EF-cells + cocktail 50% (p=0.05) Improvements in LVEF in Infarct Models Receiving ESCs Author Reference Model LV Function Results Caspi et al. JACC 2007 Rat MI Model Echo 30-60 days FS controls 20 → 10% from post MI to day 60 FS cell treated 22 → 25% (P<0.005) Author Reference Model LV Function Results VanLaake et al. Stem Cell Research 2007 Mouse MI model MRI 4 weeks , 12 weeks 4 week controls EF ~ 15%; cells EF ~ 25% (p<0.05) 12 weeks controls, cells EF ~ 18-20% (p = NS) Van Laake LW et al. 2007;Stem Cell Research 1:9-24 Van Laake LW et al. 2007;Stem Cell Research 1:9-24 Left ventricular ejection fraction (%) 80 67.6 ± 1.4 70 60.9 ± 1.4 60 50 40 hESC Saline Kearns-Jonker M, Dai W, Kloner RA Laflamme MA et al., Nature Biotechnology 2007;9:1015 In the Laflamme, Murry study, in order to obtain successful grafts of cardiomyocytes derived from human embryonic stem cells, a complex pro-survival cocktail was required in their model consisting of: 1. Activin A, bone morphogenetic protein 4 (Yields higher 2. 3. 4. 5. 6. 7. differentiation to cardiomyocytes) Matrigel A cell-permeant peptide from Bcl-XL to block mitochondrial death pathways Cyclosporine A to attenuate cyclophilin D-dependent mitochondrial pathways Pinacidil (opens KATP channels to mimic preconditioning) IGF-1 (insulin growth factor-1) to activate Akt pathways Caspase inhibitor ZVAD-fmk In Kofidis et al. study (European J Cardiothoracic Surgery 2006) 1. Allopurinal + uricase and 2. Ibuprofen pretreatment of the rats (i.e. treating the host tissue) Enhanced differentiation of transplanted hESC engraftment in mice and resulted in better restoration of myocardial function Kofidis T et al. Cardio-Thoracic Surg 2006;29:50-55 In Kloner studies & Laflamme and Murry studies: ─ Anti-apoptotic proteins did not enhance the graft (Kloner) ─ Overexpression in grafted cells of the antiapoptotic proteins Bcl-2 and Akt alone did not enhance grafts (Murry et al.) ─ Use of broad spectrum immunosupressive and antiinflammatory cocktail (including steroids) did not enhance the graft (Murry et al.) ─ In Kloner in-vitro studies, FGF (fibroblast growth factor) enhanced human embryonic graft size and myofilament content. Physiological function and transplantation of scaffold-free and vascularized human cardiac muscle tissue. KR Steven et al. PNAS 2009; 106:16568-73, 1. Patches of enriched cardiomyocytes did not demonstrate significant survival following in vivo implantation. 2. The authors created prevascularized patches from cardiomyocytes, endothelial cells (both human umbilical vein and h-ESC derived endothelial cells) and fibroblasts. 3. These vascularized patches actively contracted, could be electrically paced, and exhibited passive mechanics similar to intact myocardium. 4. Implanting these patches into in vivo skeletal muscle or heart resulted in larger viable grafts (10x) than cardiomyocytes alone. The preformed human microvessels connected with rat host coronary vasculature. 5. The authors concluded that addition of vascular and stromal elements to human myocardial grafts enhanced graft size and survivability. Tissue Engineering of Vascularized Cardiac Muscle From Human Embryonic Stem Cells Caspi O. et al. Circ. Res. 2007; 100:263-272. The authors formed synchronously contracting engineered 3D human cardiac tissue grafts formed from human embryonic stem cells with an endothelial vessel network onto a scaffold composed of a porous sponge. Addition of embryonic fibroblasts reduced endothelial cell death and improved endothelial cell proliferation. Review of studies of hES- derived cardiomyocytes implanted into rodent infarct models suggest: 1. 2. 3. 4. 5. 6. There is some degree of cell survival. Cells do thicken infarcted wall. There is improvement in LV function at least initially. EF improves initially by ~ 5 - 14%. One study showed late loss of effect. Rejection occurred in immune competent rats in our studies. Cells can be shipped over long distances and survive. Evidence of early gap junctions between transplanted cells & host. Nelson TJ et al. Repair of acute myocardial infarction by human stemness factors induced pluripotent stem cells. Circulation 2009;120:408416 Fibroblasts were transduced with stemness factors OCT 3/4, SOX 2. KLF 4 and C-MYC and converted into an embryonic stem cell-like phenotype Fibroblasts reprogrammed by stemness factors acquired the potential to repair acute myocardial infarction induced in mice Nelson TJ et al. Circulation 2009;120:408-416 Nelson TJ et al. Circulation 2009;120:408-416 Nelson TJ et al. Circulation 2009;120:408-416 Nelson TJ et al. Circulation 2009;120:408-416 iPS Programmed Without a C-MYC Yield Proficient Cardiogenesis for Functional Heart Chimerism iPS Programmed Without a C-MYC Yield Proficient Cardiogenesis for Functional Heart Chimerism. Martinez-Fernandez A, et al. Circ. Res. 2009; 105:648-656. Original nuclear reprogramming included use of oncogenes, such as cMyc to dedifferentiate the cell. The concern here is cancer. In this study transgenic expression of 3 human stemness factors SOX2, OCT4, KLF4 reset murine fibroblasts to pluripotent state. These 3factor induced pluripotent stem cells (ips) clones demonstrated cardiac differentiation including expression of cardiac markers and in vitro excitation-contraction. Thus 3-factor ips bioengineered without C-MYC could achieve cardiogenesis. We are currently studying the effects of cotransplantations of h-ESCs with MSCs alone or MSCs that are genetically engineered to overexpress heme-oxygenase. Conclusions Human - embryonic stem cell derived cardiomyocytes and induced pleuripotent stem cell-derived cardiomyocytes can form viable cardiac grafts that can contribute to improved cardiac performance. More longer term studies are needed as well as better approaches to long term cell survival. Long term data on teratomas are needed.