Introduction 2006 2004 2005 2006 2004 2001 2002 2001 2002 2006 2006 2001 2005 2006 2001 2005 2006 2004 2006 2006 2004 2006 2004 2006 2005 2004 2004 1996 2002 1998 1999 2004 2001 1996 1995 1992 1996 Materials and methods Adipose tissue donors Human subcutaneous adipose tissue samples were obtained as waste material after elective surgery and donated upon informed consent of the patients from various clinics in Amsterdam, The Netherlands. Adipose tissue was harvested from the abdomen or hip and thigh region by using either resection or tumescent liposuction. Five donors (age-range: 27–49 years) were included in this study. Isolation of the stromal vascular fraction of adipose tissue 2006 g Cell culture 5 2 2 Attachment of ASC 2 2 2 2 2 2 g Proliferation of ASC 2 2 Myocardial tissue from infarction Myocardial tissue from 27 autopsied patients who had died from AMI and from eight autopsied patients with no cardiac lesions of any kind was obtained from the Department of Pathology for authopsy, with approval of the ethics committee of the VU Medical Centre, Amsterdam. The use of left-over material after the pathological examination is completed, is part of the standard patient contract in our hospital. Heart tissue was sampled as soon as possible, within 24 h of death. Characterization of infarction phase 2003 1 Table 1 LDH Infarctionphase Numberof patients Infarct duration Definition 0 8 No infarction Does not apply 1 11 3–12 h LDH decolourization,without extravascularneutrophilic granulocytes. 2 9 12 h–5 days Extravascular neutrophilicgranulocytes. 3 7 5–14 days Granulation tissueformation Immunohistochemistry 2003 2 2 For fibronectin staining, antigen retrieval was performed by incubating the sections with 0.1% pepsine-HCl for 30 min at 37°C. Sections were incubated with rabbit antibodies against human fibronectin (1:18,000) for 1 h at room temperature, followed by incubation with 100 μl envision (ready for use kit, anti-Mouse IgG and anti-Rabbit IgG) for 30 min at room temperature. Staining was visualized with envision DAB (DakoCytomation). Finally, the sections were counterstained with haematoxylin, dehydrated and cover-slipped. Control sections were incubated with PBS instead of the primary antibody. All slides were judged and scored for infarct age and anatomical localization of fibronectin and C3d, as visualized by immunohistochemical staining. The border-zone of the infarct was defined as the area surrounding the microscopic infarction area, as characterized by C3d staining. Since only the fibronectin on the plasma membrane and in the ECM are available for ASC attachment, fibronectin deposition was scored separately for the ECM, plasma membrane and intracellular components (nucleus and cytoplasm). The extent of the fibronectin deposits was determined by quantifying the mean surface area occupied by cardiomyocytes positive for fibronectin and expressed as a percentage of the total surface area of the sections in the infarcted region, border-zone and non-infarcted areas. Areas were scored as negative (0), 1%–5% of the cells positive (1), 6%–25% positive (2), 26%–50% positive (3), 51%–75% positive (4) or 76%–100% positive (5). Since positivity of fibronectin in the ECM is difficult to quantify, fibronectin staining in ECM was related to its intensity score as follows: negative (0), minor positive (1), intermediate positive (2) or strongly positive (3). Statistics P Results Attachment and proliferation of ASCs on fibronectin 1 P 2 2 Fig. 1 col-1 fibro 2 a b c Error bars n P  2 P 2 2 Fig. 2 2 a b c Error bars n P  P n 3 Fig. 3 2  Fibronectin accumulation after infarction 4 4 4 5 P P Fig. 4 a arrow I arrow II arrow III b arrow IV c d c  Fig. 5 Error bars P P  P P P 6 6 6 Fig. 6 IH a b c Error bars n n n n n  Discussion P P n 2 2 2005 2004 2005 2004 In an experiment mimicking the in vivo situation after a myocardial infarction, we have demonstrated that fibronectin also increases the attachment of ASC to cardiac muscle cells, even when a relatively low concentration of fibronectin is used. These results are in agreement with our hypothesis that fibronectin depositions will also increase stem cell attachment in vivo, independent of fibronectin concentration, underlining the importance of fibronectin in stem cell transplantation. P 2005 Since the presence of fibronectin promotes ASC attachment and proliferation in vitro in our study, we have hypothesized that the presence of fibronectin in the heart after myocardial infarction might positively affect stem cell adhesion and proliferation at the site of injury. As such, stem cell therapy would be most efficient when fibronectin depositions are high. We have found an increase in the extent of fibronectin depositions in cardiomyocytes during AMI phase 2, whereas fibronectin deposition in the ECM is significantly increased in the infarction area during AMI phase 2 and phase 3, when compared with the uninfarcted heart. 1997 1995 1992 1990 1997 1996 2000 2003 2000 1996 2000 2006 2006 2004 2005 In conclusion, we have shown, for the first time, that fibronectin improves ASC attachment and proliferation. To achieve a positive effect of fibronectin on stem cell therapy after AMI in the heart, this therapy should be applied when fibronectin depositions in the heart are high. We have accurately scored fibronectin deposition in the human heart after AMI and found that the fibronectin intensity score significantly increases from 12 h after MI in both the infarction area and the border-zone.