The in vitro culture of porcine bone marrow-derived mesenchymal stem cells (MSCs) was used for the investigation of adult stem cell biology. Isolated porcine MSCs possessed the ability to proliferate extensively in an antioxidants-rich medium containing 5% fetal bovine serum (FBS). Greater than 40 serial MSC passages and 100 cell population doublings have been recorded for some MSC batches. Early and late passage MSCs were defined here as those cultures receiving less than 5 trypsin passages and more than 15 trypsin passages, respectively. Consistent with their robust ability to proliferate, both the early and late passage MSCs expressed the cell-cycle promoting enzyme p34cdc2 kinase. Late MSCs, however, exhibited certain features reminiscent of cellular aging such as actin accumulation, reduced substrate adherence, and increased activity of lysosomal acid beta-galactosidase. Early MSCs retained the multipotentiality capable of chondrogenic, osteogenic, and adipogenic differentiation upon induction in vitro. In contrast, late MSCs were only capable of adipogenic differentiation, which was greatly enhanced at the expense of the osteochondrogenic potential. Along with these changes in multipotentiality, late MSCs expressed decreased levels of the bone morphogenic protein (BMP-7) and reduced activity of alkaline phosphatase. Late MSCs also exhibited attenuated synthesis of the hematopoietic cytokines granulocyte colony-stimulating factor (G-CSF), leukemia inhibitory factor (LIF), and stem cell factor (SCF). The long-term porcine MSC culture, thus, provides a model system to study the molecular interplay between multiple MSC differentiation cascades in the context of cellular aging.

Heart failure carries a poor prognosis with few treatment options. While myocardial stem cell therapeutic trials have traditionally relied on intracoronary infusion or intramyocardial injection routes, these cell delivery methods are invasive and can introduce harmful scar tissue, arrhythmia, calcification, or microinfarction in the heart. Given that patients with heart failure are at an increased surgical risk, the development of a noninvasive stem cell therapeutic approach is logistically appealing. Taking advantage of the trophic effects of bone marrow mesenchymal stem cells (MSCs) and using a hamster heart failure model, the present study demonstrates a novel noninvasive therapeutic regimen via the direct delivery of MSCs into the skeletal muscle bed. Intramuscularly injected MSCs and MSC-conditioned medium each significantly improved ventricular function 1 mo after MSC administration. MSCs at 4 million cells/animal increased fractional shortening by approximately 40%, enhanced capillary and myocyte nuclear density by approximately 30% and approximately 80%, attenuated apoptosis by approximately 60%, and reduced fibrosis by approximately 50%. Myocyte regeneration was evidenced by an approximately twofold increase in the expression of cell cycle markers (Ki67 and phosphohistone H(3)) and an approximately 13% reduction in mean myocyte diameter. Increased circulating levels of hepatocyte growth factor (HGF), leukemia inhibitory factor, and macrophage colony-stimulating factor were associated with the mobilization of c-Kit-positive, CD31-positive, and CD133-positive progenitor cells and a subsequent increase in myocardial c-Kit-positive cells. Trophic effects of MSCs further activated the expression of HGF, IGF-II, and VEGF in the myocardium. The work highlights a cardiac repair mechanism mediated by trophic cross-talks among the injected MSCs, bone marrow, and heart that can be explored for noninvasive stem cell therapy.

Calpain-mediated TnI proteolysis can be dissociated from stunning and arises from elevations in preload rather than ischemia. This raises the possibility that ongoing preload-induced TnI degradation could impair myocardial function long-term.

Abstract-Fibroblast growth factors (FGFs) have diverse actions on the myocardium but the importance of stimulating angiogenesis versus direct effects of FGFs on cardiac myocytes is unclear. We used intracoronary injection of a replication-deficient adenoviral construct overexpressing FGF-5 (AdvFGF-5) to improve flow and function in swine with hibernating myocardium. Two-weeks after AdvFGF-5 (nϭ8), wall-thickening increased from 2.4Ϯ0.04 to 4.7Ϯ0.7 mm in hibernating LAD regions (PϽ0.05) whereas remote wall-thickening was unchanged (6.7Ϯ0.4 to 5.8Ϯ0.5 mm). This was associated with small increases in resting flow to dysfunctional myocardium, but flow during adenosine was unchanged (LAD 1.45Ϯ0.27 versus 1.46Ϯ0.23 mL/min per g and remote 4.84Ϯ0.23 versus 4.71Ϯ0.47 mL/min per g, PϭNS). Unexpectedly, animals receiving AdvFGF-5 demonstrated a 29% increase in LV mass over the 2-week period (PϽ0.05 versus untreated animals with hibernating myocardium and normal shams). Histological analysis confirmed profound myocyte cellular hypertrophy in AdvFGF-5 treated myocardium (19.9Ϯ0.32 versus 15.2Ϯ0.92 m in untreated, PϽ0.001). Myocytes in the proliferative phase of the cell cycle (Ki-67 staining) increased 7-fold after AdvFGF-5 (2,904Ϯ405 versus 409Ϯ233 per 10 6 myocyte nuclei in untreated, PϽ0.05). Myocyte nuclei in the mitotic phase (phosphorylated histone H3 staining) also increased after myocyte nuclei in untreated, PϽ0.05). Thus, rather than angiogenesis, stimulation of hypertrophy and reentry of a small number of myocytes into the mitotic phase of the cell cycle are responsible for the effects of AdvFGF-5 on function. Although additional mechanisms may contribute to the improvement in wall-thickening, overexpression of AdvFGF-5 may afford a way to restore function in hibernating myocardium and ameliorate heart failure in chronic ischemic cardiomyopathy. Key Words: hibernating myocardium Ⅲ growth factors Ⅲ gene therapy H ibernating myocardium is common in patients with ischemic cardiomyopathy, and myocardial revascularization can improve function and ameliorate symptoms of heart failure. Unfortunately, many patients are not suitable candidates for surgical or percutaneous revascularization and developing nonsurgical approaches to reverse dysfunction and improve perfusion would be desirable. Administration of FGFs as recombinant proteins or overexpression using plasmid and adenoviral vectors elicits multiple effects that could favorably affect flow and function in viable chronically dysfunctional myocardium. Considerable enthusiasm for therapeutic angiogenesis has arisen from promising experimental animal studies using rapidly developing coronary collaterals and ameroid occluder models. [1][2][3][4][5][6] Unfortunately, FGF-mediated improvements in myocardial perfusion are small, and few laboratories have demonstrated objective changes in flow during pharmacological or metabolic stress. In addition, when administered to dogs with chronic welldeveloped coronary collaterals, FGF did not improve myocardial perfusion. 5 This ...

Rationale: Mesenchymal stem cells (MSCs) improve function after infarction, but their mechanism of action remains unclear, and the importance of reduced scar volume, cardiomyocyte proliferation, and perfusion is uncertain.Objective: The present study was conducted to test the hypothesis that MSCs mobilize bone marrow progenitor cells and improve function by stimulating myocyte proliferation in collateral-dependent hibernating myocardium. Methods and Results: Swine with chronic hibernating myocardium received autologous intracoronary MSCs (icMSCs; Ϸ44؋106 cells, n‫)01؍‬ 4 months after instrumentation and were studied up to 6 weeks later. Physiological and immunohistochemical findings were compared with untreated hibernating animals (n‫,)7؍‬ sham-normal animals (n‫,)5؍‬ and icMSC-treated sham-normal animals (n‫. Key Words: mesenchymal stem cells Ⅲ hibernating myocardium Ⅲ bone marrow progenitor cells M esenchymal stem cells (MSCs) can repair a variety of tissues after injury 1 and are currently being used in clinical trials to treat patients with cardiovascular disease. 2 Nevertheless, their precise mechanism of action and the role of myocyte regeneration versus angiogenesis are controversial. Most preclinical investigation has focused on animal models of myocardial infarction in which intravenous, intramyocardial, and intracoronary administration of MSCs have been demonstrated to reduce infarct size and improve left ventricular (LV) function in both acute and chronic infarction. [3][4][5][6][7][8][9][10][11][12][13] Although early studies demonstrated the ability of autologous and allogeneic MSCs to differentiate into vessels, 8,14 smooth muscle, 14 and cardiac muscle, 15,16 the quantitative extent of MSC differentiation into a cardiac cellular phenotype has been small and inconsistent with measured reductions in infarct volume. 4,8,11 Recent investigations have hypothesized that MSCs effect cardiac repair through a paracrine mechanism that stimulates proliferation of endogenous myocytes, but in vivo studies quantifying the magnitude of myocyte proliferation are limited. Most analyses focus on the small rim of border zone tissue between normal and infarcted myocardium rather than larger remote regions or areas at risk of ischemia. 5,9,[17][18][19][20][21][22] Recently, we demonstrated that pravastatin can mobilize cKit ϩ and CD133 ϩ bone marrow progenitor cells (BMPCs). 23 The BMPCs localized in the heart and were accompanied by improved myocardial function in swine, with hibernating myocardium devoid of infarction in the absence of an increase in myocardial perfusion. Although BMPC mobilization had no effect on myocyte proliferation in the normal heart, it increased the frequency of myocytes in the proliferative phase of the cell cycle in diseased hearts and increased myocyte nuclear density with small myocytes that suggested that endogenous myocyte proliferation was stimulated. 23 Likewise, studies with skeletal muscle injection of porcine MSCs into Syrian hamsters 24 and MSCs that overexpressed insulin-like ...