ARTICLES Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, 75 Francis Street, Boston, Massachusetts, 02115 USA. Correspondence should be addressed to V.J.D. (vdzau@partners.org). Published online 10 August 2003; doi:10.1038/nm912 NATURE MEDICINE VOLUME 9 | NUMBER 9 | SEPTEMBER 2003 1195 Prolonged interruption of myocardial blood flow initiates events that culminate in cardiac myocyte death 1 . Proposed endogenous reparative mechanisms include cardiac myocyte hypertrophy 2 and hyperplasia 3 and the trafficking of bone marrow–derived stem cells (BMCs) to the myocardium for repair and angiogenesis 4,5 . None of these proposed mechanisms are adequate in restoring lost myocardium or sustaining cardiac function. Harvesting BMCs followed by direct injection into ischemic myocardium results in angiogenesis and myogenesis 6–8 , but functional improvement is incomplete. It is unclear whether trans- plantation of BMCs affects repair of the ischemic myocardium pri- marily by angioblast-mediated vasculogenesis 9,10 , which prevents apoptosis of native cardiac myocytes, or by direct regeneration of the lost myocytes. Mesenchymal stem cells (MSCs) are self-renewing, clonal pre- cursors of non-hematopoietic tissues. They are expandable in culture and multipotent, and can differentiate into osteoblasts 11 , chondro- cytes 11 , astrocytes 12 , neurons 13 and skeletal muscle 14 . Several groups have reported that putative MSCs derived from bone marrow can dif- ferentiate into cardiac muscle in vitro 15 and in vivo 16,17 . However, it has been observed that transplantation of as many as 6 × 10 7 of these puta- tive MSCs into infarcted porcine hearts yielded only marginal improvement in cardiac function 17 . This is explained at least in part by poor viability of the transplanted cells. It has been estimated that >99% of MSCs die 4 d after transplantation into uninjured nude- mouse hearts 16 . Cell transplantation strategies to replace lost myocardium are limited by the inability to deliver large numbers of cells that resist peritransplantation graft cell death 17–20 . Accordingly, we set out to isolate and expand a highly purified pop- ulation of adult rat bone marrow–derived MSCs, and to engineer these cells to overexpress Akt, a serine threonine kinase and powerful sur- vival signal in many systems 21,22 , to test the hypothesis that Akt- engineered MSCs are more resistant to apoptosis and can enhance cardiac repair after transplantation into the ischemic rat heart. Our results documented significant retention of Akt-MSCs in the ischemic heart that was associated with inhibition of cardiac remodeling, a greater volume of regenerated myocardium, and near-complete normalization of systolic and diastolic cardiac function. RESULTS MSCs express markers distinct from hematopoietic stem cells MSCs proliferated in mixed culture with hematopoietic cells, yielding 5 × 10 6 cells by day 15 of culture. We separated MSCs from hematopoi- etic cells based on their preferential attachment to polystyrene sur- faces 23 . By immunocytochemistry, over 99% of MSCs expressed CD29, CD71, CD90, CD106 and CD117 (Fig. 1); 60% expressed Ki67 and 15% expressed the transcription factors Nkx2.5 (Fig. 1) and Gata-4 (data not shown). MSCs did not express the hematopoietic markers CD34 and CD45 (Fig. 1) or the cardiac-specific markers myosin heavy chain (MHC), myosin light chain (MLC), cardiac troponin I (CTnI), α-sarc- omeric-actin (αSA) or MEF-2 (data not shown). In addition, MSCs also expressed the gap junction protein connexin-43 as verified by RT- PCR (data not shown). We further purified MSCs using negative para- magnetic bead sorting targeting CD34, resulting in a population that was >99.9% pure.We were unable to induce these MSCs to differentiate into megakaryocytes and erythroid cells using described methods 24 . Increased Akt activity protects against MSC apoptosis We used retroviruses to transduce MSCs with genes expressing green Mesenchymal stem cells modified with Akt prevent remodeling and restore performance of infarcted hearts Abeel A Mangi, Nicolas Noiseux, Deling Kong, Huamei He, Mojgan Rezvani, Joanne S Ingwall & Victor J Dzau Transplantation of adult bone marrow–derived mesenchymal stem cells has been proposed as a strategy for cardiac repair following myocardial damage. However, poor cell viability associated with transplantation has limited the reparative capacity of these cells in vivo. In this study, we genetically engineered rat mesenchymal stem cells using ex vivo retroviral transduction to overexpress the prosurvival gene Akt1 (encoding the Akt protein). Transplantation of 5 × 10 6 cells overexpressing Akt into the ischemic rat myocardium inhibited the process of cardiac remodeling by reducing intramyocardial inflammation, collagen deposition and cardiac myocyte hypertrophy, regenerated 80–90% of lost myocardial volume, and completely normalized systolic and diastolic cardiac function. These observed effects were dose (cell number) dependent. Mesenchymal stem cells transduced with Akt1 restored fourfold greater myocardial volume than equal numbers of cells transduced with the reporter gene lacZ. Thus, mesenchymal stem cells genetically enhanced with Akt1 can repair infarcted myocardium, prevent remodeling and nearly normalize cardiac performance. © 2003 Nature Publishing Group http://www.nature.com/naturemedicine