Cardiac Mesenchymal Stem Cells Research Articles

Defective Myofibroblast Formation from Mesenchymal Stem Cells in the Aging Murine Heart: Rescue by Activation of the AMPK Pathway

Aged mice in a murine model of myocardial infarction exhibit less effective myocardial repair. We hypothesized that the deficiency arises from altered lineage choice of endogenous mesenchymal stem cells (MSCs) and faulty maturation of myofibroblasts. Examination of cardiac MSCs revealed a substantial reduction in the pluripotency marker Nanog in cells from aged mice. In addition, the aged MSCs demonstrated a redirected lineage choice that favored adipocytic commitment over fibroblast or myofibroblast differentiation. Fibroblasts derived from aged MSCs demonstrated reduced expression of transforming growth factor-β (TGF-β) receptors I and II and diminished SMAD3 phosphorylation, associated with attenuated contractility and migration. Overexpression of constitutively active TGF-β receptor I in aged cardiac fibroblasts ameliorated their defective motility but did not improve their contractility. Culturing of MSCs and fibroblasts with AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside) to activate adenosine monophosphate-activated kinase resulted in TGF-β-dependent development of myofibroblasts with markedly enhanced contractility despite no reduction in adipocytic commitment or increased expression of TGF-β receptors and SMAD3 phosphorylation. The data suggest an adenosine monophosphate-activated kinase-dependent gain of function as mediated by phosphorylation of TGF-β-activated kinase 1 and p38 mitogen-activated protein kinase, which amplifies the response to TGF-β1 via a non-canonical pathway, thus compensating for the reduced expression of TGF-β receptors.

Abstract 1015: Human Heart Contains A Stem Cell Population With Mesenchymal Properties

Background Mesenchymal Stem Cells (MSC) were initially isolated from the bone marrow (BM) and identified as plastic adherent cells with a typical immunophenotype (negative for CD45, CD34, CD117 and HLA-DR and positive for CD105, CD90, CD44, CD73 and CD13), and able to differentiate into mesodermal lineages in vitro . Recently, MSC have been isolated from different adult tissues, i.e. liver, fat, kidney and thymus. Aim of the present study was to search for Cardiac Mesenchymal Stem Cells (C-MSC) in the adult human heart. Methods and Results : Auricle fragments were obtained from adult patients undergoing cardiac surgery. From these specimens we isolated a high proliferating plastic adherent cell population that, by FACS analysis, expressed mesenchymal markers (CD105, CD44, CD73, CD13 ) and was negative for hematopoietic markers (CD45, CD34) and HLA-DR. Further, in appropriate differentiating media, these cells exhibited osteogenic and adipogenic differentiation as cultures were positive for Oil Red O and Von Kossa staining used to mark intracellular lipid droplets and calcium salts deposits, respectively. Quantitative Real Time (qRT) -PCR analysis revealed that cells were negative for the early cardiac markers Nkx2.5 and Tbx5 and for the adult cardiac markers α-Myosin Heavy Chain and Myosin Light Chain-2a. Interestingly, in standard culture conditions, cells were positive for the early cardiac marker GATA4; in contrast, BM-MSC expressed GATA4 only after dexamethasone treatment. Additionally, when injected into the zebrafish blastula (about 100 –200 cells, n=70), C-MSC specifically homed in the cardiac region, as found at 24 –28 hpf (hours post fertilization). In contrast, transplanted BM-MSCs, were found not only in proximity of the heart, but throughout several mesodermal derivatives of the embryo (n=25). Conclusions: The adult human heart contains C-MSCs that can be easily expanded in vitro and may represent a useful population for autologous cell therapy of some cardiac diseases.

Abstract 1855: Transmural Myocardial Repair with Engineered Heart Tissue Grafts

Engineered Heart Tissue (EHT) can be utilized to partially repair infarcted myocardium in rats. Here, we investigated the feasibility of EHT-grafts as transmural wall replacement in a heterotopic transplantation model. Methods: EHTs (diameter: 15 mm, thickness: 1– 4 mm) were generated from 12.5 ×10 6 neonatal rat heart cells, collagen type I, and matrigel. Similarly, non-contractile constructs were generated from rat cardiac fibroblasts (FB) and mesenchymal stem cells (MSC). Grafts were surgically inserted into large transmural defects (diameter: 6 mm) in the left ventricle of explanted donor hearts. Subsequently, “treated” hearts were transplanted into weight-matched (308±12 g; n=14), immune suppressed (cyclosporine, azathioprine, prednisolone) Wistar rats in heterotopic position. All transmural defects were also covered with an aortic patch to prevent bleeding from the ventricles. Sham surgeries included aortic patch implantations only. Heterotopic hearts were harvested after 28 days and subjected to morphological analyses by confocal laser scanning microscopy (CLSM). Results: Heart transplant weight at the time of implantation was 1.1±0.02 g (n=14). Heterotopic heart weight increased substantially in Sham (2.4±0.3 g, n=3) and FB-graft (2.1±0.1 g, n=3) animals, whereas MSC- (1.7±0.2 g, n=4) and EHT-graft (1.3±0.1 g, n=4; p<0.05 vs. Sham) animals showed a smaller or no increase in weight, respectively. EHT grafts remained contractile throughout the observation period. CLSM revealed that EHT-grafts established oriented muscle bundles (actin and actinin staining) inside the transmural defects and were strongly vascularized (CD31 and smooth muscle actin staining; lectin labeling) leading to partial reconstitution of the myocardial continuity. This was not observed in animals with FB- and MSC-grafts. However, MSC-grafts, but not FB-grafts, contained newly formed vessels with a markedly larger diameter than observed in EHT-grafts (21±6 vs. 5±0.7 μm; p<0.05). Conclusion: EHTs can be utilized as myocardial tissue grafts to reconstruct and prevent pathological enlargement of the left ventricle. This study constitutes a first step to establish a novel transmural myocardial repair technology involving fully bioengineered heart muscle.

Abstract 1016: Regenerative Cellular Therapy: How To Enrich The Mesenchymal Stem Cell Content In The Periinfarction Zone After Myocardial Infarction

Background: The pluripotent capacity of adult mesenchymal stem cells (MSCs) makes this cell type a potential candidate for tissue regeneration after myocardial infarction (MI). However, the most effective route of MSC application and the impact of contributory cytokine enhancement remains to be elucidated. Methods and Results: Murine CD90 + CD45-CD34-sca-1 + c-kit-MSCs were culture-expanded, stably transfected with firefly luciferase for bioluminescence imaging (BI), and labeled with CSFE for histology (H). High-throughput proteomics and a battery of in vitro and in vivo assays were used to profile 20 key cardiac-specific cytokines upregulated following MI for their effects on the biologic performance of MSCs. VEGF and HGF showed most effective enhancement of MSC proliferation, migration, and trans-membrane invasion. After LAD ligation, syngeneic mice received intraperitoneal (IP), intravenous (IV) or intramyocardial (IC) MSC injections. The development of cardiac MSC content was closely monitored by in vivo BI and H. Cytokine enhancement (CE) protocol: VEGF 8ng/ml and HGF 40ng/ml were injected into the infarction border zone to attract MSCs to the heart (after IV- or IP-injection) or to retain them in the heart (after IC-injection). 1 day after IP injection, a maximum of only 0.4 ± 0.5 cells/HPF was detected in the heart. Most IP-injected MSCs failed to be absorbed and enter the circulation. They rapidly decayed to <10% within the peritoneum over 4 ± 2 days. Due to their large size (19um), huge amounts of IV-injected MSCs were trapped within the pulmonary capillaries, causing hemodynamic alterations if >10 6 cells were used. After 1 day, a maximum of 1 ± 0.5 cells/HPF was found in the heart and CE significantly increased the cell content to 6 ± 3 cells/HPF (p=0.020). IC-injected MSCs vanished rapidly and cell counts dropped to 32 ± 18% at 6 days and 9 ± 6% at 12 days. Interestingly, the CE protocol significantly prolonged intramyocardial MSC survival and preserved higher cell counts (125 ± 21% at 6 days, p<0.001 and 87 ± 31% at 12 days, p<0.001). Conclusions: The most effective route of MSC application seems to be direct injection into the heart combined with cytokine enhancement in the border zone to create a beneficial microenviroment for cell survival.

Introduction

Introduction