Impact Of Donor Age On The Efficacy Of Cell Therapy For Myocardium Infarction

Objective: Autologous cell transplantation for the treatment of myocardial dysfunction is undergoing clinical trials. Although animal models have provided evidence for the efficacy of cellular transplantation from young and otherwise healthy animals, the impact of cell donor age remains to be elucidated. Methods: In vitro study Growth characteristics and proliferative capacity of vascular smooth muscle (SMC), bone marrow cells (BMC), and skeletal myoblasts (SKMC) from different age groups were compared in rats. Freshly isolated SMCs, BMCs and SKMC were counted and 1 × 10~4 cells from 6 rats from each group were seeded into 5 cm dishes. Cell number at 1, 2, 3 and 4 weeks of culture was counted and cell number of two dishes/time point/animal/cell type was averaged. Numbers of mesenchymal and hematopoietic stem cells (MSC and HSC) in BMCs were estimated using Cloning Forming Unit (CFU) and CFU-Granulocyte-Macrophage (GM) assays, respectively. Myogenic potential of MSC was estimated by desmin staining after treatment with 5-azacytidine. The purity of SMC was estimated by SMC- a-actin.In vivo study Syngeneic rats underwent proximal left coronary artery ligation. Three weeks later, 3.5 million SMC (n=11), BMC (n=9) from 2 year old donor rats or culture medium (n=6) were injected in the infarct region. Five weeks after implantation cardiac function was assessed by echocardiography. And then the hearts were quickly excised and perfused in a Langendorff apparatus. Peak systolic pressure (PSP) and end diastolic pressure (EDP) were measured.. The infarcted areas were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). To identify the transplanted cells, the sections were also incubated with the antibody against BrdU. To follow up the differentiation of the grafted BMCs, the polyclonal antibody against sarcomeric myosin heavy chains (MHC) were used for immunochemical staining. To measure vascular density, three specimens stained with H&E from each group were randomly selected. Five fields in each section were randomly selected and their vascular densities were counted and expressed as blood vessels/0.2mm~2 by a blinded observer at 200X magnification.