Techung Lee

Phenotypic changes of adult porcine mesenchymal stem cells induced by prolonged passaging in culture.

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 β‐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.

Preload Induces Troponin I Degradation Independently of Myocardial Ischemia

BackgroundAlthough global ischemia induces troponin I (TnI) degradation, regional ischemia does not. We hypothesized that this disparity is related to preload-induced proteolysis, which varies as a function of the amount of myocardium at risk of ischemia. Methods and ResultsIsolated rat hearts were buffer-perfused at controlled levels of preload. Increasing preload to 25 mmHg in the absence of ischemia produced pronounced TnI degradation (27 kDa versus 31 kDa bands: 16.4±3.6% versus 4.7±1.9% in immediately excised controls, P <0.05). TnI degradation could be blocked by preventing the activation of endogenous calpains with 25 &mgr;mol/L calpeptin (4.3±0.6%). This improved function, with left ventricular systolic pressure increasing from 103±4 mmHg to 137±7 mmHg (P <0.05). Eliminating elevations in preload after global ischemia-induced stunning also prevented TnI degradation. ConclusionsCalpain-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.

Heart failure therapy mediated by the trophic activities of bone marrow mesenchymal stem cells: a noninvasive therapeutic regimen

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.

Vascular endothelial growth factor (VEGF) as a key therapeutic trophic factor in bone marrow mesenchymal stem cell-mediated cardiac repair

We recently demonstrated a novel effective therapeutic regimen for treating hamster heart failure based on injection of bone marrow mesenchymal stem cells (MSCs) or MSC-conditioned medium into the skeletal muscle. The work highlights an important cardiac repair mechanism mediated by the myriad of trophic factors derived from the injected MSCs and local musculature that can be explored for non-invasive stem cell therapy. While this therapeutic regimen provides the ultimate proof that MSC-based cardiac repair is mediated by the trophic actions independent of MSC differentiation or stemness, the trophic factors responsible for cardiac regeneration after MSC therapy remain largely undefined. Toward this aim, we took advantage of the finding that human and porcine MSCs exhibit species-related differences in expression of trophic factors. We demonstrate that human MSCs when compared to porcine MSCs express and secrete 5-fold less vascular endothelial growth factor (VEGF) in conditioned medium (40+/-5 and 225+/-17 pg/ml VEGF, respectively). This deficit in VEGF output was associated with compromised cardiac therapeutic efficacy of human MSC-conditioned medium. Over-expression of VEGF in human MSCs however completely restored the therapeutic potency of the conditioned medium. This finding indicates VEGF as a key therapeutic trophic factor in MSC-mediated myocardial regeneration, and demonstrates the feasibility of human MSC therapy using trophic factor-based cell-free strategies, which can eliminate the concern of potential stem cell transformation.

Adenoviral Gene Transfer of FGF-5 to Hibernating Myocardium Improves Function and Stimulates Myocytes to Hypertrophy and Reenter the Cell Cycle

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 (n=8), wall-thickening increased from 2.4±0.04 to 4.7±0.7 mm in hibernating LAD regions (P<0.05) whereas remote wall-thickening was unchanged (6.7±0.4 to 5.8±0.5 mm). This was associated with small increases in resting flow to dysfunctional myocardium, but flow during adenosine was unchanged (LAD 1.45±0.27 versus 1.46±0.23 mL/min per g and remote 4.84±0.23 versus 4.71±0.47 mL/min per g, P=NS). Unexpectedly, animals receiving AdvFGF-5 demonstrated a 29% increase in LV mass over the 2-week period (P<0.05 versus untreated animals with hibernating myocardium and normal shams). Histological analysis confirmed profound myocyte cellular hypertrophy in AdvFGF-5 treated myocardium (19.9±0.32 versus 15.2±0.92 &mgr;m in untreated, P<0.001). Myocytes in the proliferative phase of the cell cycle (Ki-67 staining) increased 7-fold after AdvFGF-5 (2,904±405 versus 409±233 per 106 myocyte nuclei in untreated, P<0.05). Myocyte nuclei in the mitotic phase (phosphorylated histone H3 staining) also increased after AdvFGF-5 (127±24 versus 35±13 per 106 myocyte nuclei in untreated, P<0.05). 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.

Autologous Mesenchymal Stem Cells Mobilize cKit+ and CD133+ Bone Marrow Progenitor Cells and Improve Regional Function in Hibernating Myocardium

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; ≈44×106 cells, n=10) 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=6). In hibernating myocardium, icMSCs increased function (percent wall thickening of the left anterior descending coronary artery 24±4% to 43±5%, P<0.05), although left anterior descending coronary artery flow reserve (adenosine/rest) remained critically impaired (1.2±0.1 versus 1.2±0.1). Circulating cKit+ and CD133+ bone marrow progenitor cells increased transiently after icMSC administration, with a corresponding increase in myocardial cKit+/CD133+ and cKit+/CD133− bone marrow progenitor cells (total cKit+ from 223±49 to 4415±866/106 cardiomyocytes, P<0.05). In hibernating hearts, icMSCs increased Ki67+ cardiomyocytes (from 410±83 to 2460±610/106 nuclei, P<0.05) and phospho-histone H3–positive cardiomyocytes (from 9±5 to 116±12/106 nuclei, P<0.05). Myocyte nuclear number (from 75 336±5037 to 114 424±9564 nuclei/mm3, P<0.01) and left ventricular mass (from 2.5±0.1 to 2.8±0.1 g/kg, P<0.05) increased, yet myocytes were smaller (14.5±0.4 versus 16.5±0.4 &mgr;m, P<0.05), which supports endogenous cardiomyocyte proliferation. In sham-normal animals, icMSCs increased myocardial bone marrow progenitor cells with no effect on myocyte proliferation or regional function. Conclusions: Our results indicate that icMSCs improve function in hibernating myocardium independent of coronary flow or reduced scar volume. This arises from stimulation of myocyte proliferation with increases in cKit+/CD133+ bone marrow progenitor cells and cKit+/CD133− resident stem cells, which increase myocyte number and reduce cellular hypertrophy.

Absence of Troponin I Degradation or Altered Sarcoplasmic Reticulum Uptake Protein Expression After Reversible Ischemia in Swine

The findings of troponin I (TnI) proteolysis (in isolated rat hearts) and induction of selected sarcoplasmic reticulum (SR) calcium-regulatory genes (after repetitive total coronary occlusions in swine) have given rise to the hypothesis that the time course of functional recovery of stunned myocardium reflects the resynthesis of reversibly damaged proteins. Although stunning occurs after brief total occlusions and prolonged partial occlusions (ie, short-term hibernation), the time course of functional recovery varies from a few hours to several days, suggesting that the severity of protein damage or mechanisms responsible for the dysfunction may differ. To study this, we examined SR gene expression and TnI degradation in stunned myocardium produced by 10-minute total left anterior descending coronary artery (LAD) occlusions (n=4) or 1-hour partial LAD occlusions, in which flow was reduced to approximately 50% of control values for 60 minutes (n=6) in swine. One hour after reperfusion, LAD wall thickening was severely depressed in both models despite normal perfusion and no triphenyltetrazolium chloride evidence of necrosis. Normal myocardium exhibited TnI immunoreactivity at 31 kDa and a weak secondary band at 27 kDa. Irreversible injury or calpain activation in vitro produced a marked increase in the intensity of the 27-kDa band, consistent with TnI degradation. Stunned myocardium demonstrated no change in the 31- or the 27-kDa band, and the percentage of the 27- to 31-kDa band remained constant after 10-minute total occlusions (LAD, 5.9+/-0.9%; normal, 4.9+/-1.6%) and 1-hour partial occlusions (LAD, 8.5+/-1.9%; normal, 7.3+/-1.4%) and in sham controls (LAD, 10.9+/-1.5%; normal, 9.8+/-1.4%). Northern analysis showed no alterations in TnI or SR gene expression, but the stress protein HSP-70 was variably induced. Thus, stunned myocardium occurs without TnI degradation or altered SR gene expression, indicating that additional mechanisms are responsible for the reversible dysfunction after single episodes of regional ischemia in swine.

Activation of host tissue trophic factors through JAK-STAT3 signaling: a mechanism of mesenchymal stem cell-mediated cardiac repair

We recently demonstrated a cardiac therapeutic regimen based on injection of bone marrow mesenchymal stem cells (MSCs) into the skeletal muscle. Although the injected MSCs were trapped in the local musculature, the extracardiac cell delivery approach repaired the failing hamster heart. This finding uncovers a tissue repair mechanism mediated by trophic factors derived from the injected MSCs and local musculature that can be explored for minimally invasive stem cell therapy. However, the trophic factors involved in cardiac repair and their actions remain largely undefined. We demonstrate here a role of MSC-derived IL-6-type cytokines in cardiac repair through engagement of the skeletal muscle JAK-STAT3 axis. The MSC IL-6-type cytokines activated JAK-STAT3 signaling in cultured C2C12 skeletal myocytes and caused increased expression of the STAT3 target genes hepatocyte growth factor (HGF) and VEGF, which was inhibited by glycoprotein 130 (gp130) blockade. These in vitro findings were corroborated by in vivo studies, showing that the MSC-injected hamstrings exhibited activated JAK-STAT3 signaling and increased growth factor/cytokine production. Elevated host tissue growth factor levels were also detected in quadriceps, liver, and brain, suggesting a possible global trophic effect. Paracrine actions of these host tissue-derived factors activated the endogenous cardiac repair mechanisms in the diseased heart mediated by Akt, ERK, and JAK-STAT3. Administration of the cell-permeable JAK-STAT inhibitor WP1066 abrogated MSC-mediated host tissue growth factor expression and functional improvement. The study illustrates that the host tissue trophic factor network can be activated by MSC-mediated JAK-STAT3 signaling for tissue repair.

Chemical hypoxia triggers apoptosis of cultured neonatal rat cardiac myocytes: modulation by calcium-regulated proteases and protein kinases.

Myocardial infarctions and stroke arise primarily as a result of hypoxia/ischemia-induced cell injury. However, the molecular mechanism of cardiac cell death due to hypoxia has not been elucidated. We showed here that chemical hypoxia induced by 1 mM azide triggered apoptosis of isolated neonatal rat ventricular cardiac myocytes but had no effect on cardiac fibroblasts. The azide-induced cardiomyocyte apoptosis could be characterized by a reversible initiation phase (0-6 h after azide exposure) during which cytosolic ATP levels remained little affected. This was followed by an irreversible execution phase (12-18 h) exhibiting prominent internucleosomal DNA fragmentation, cell membrane leakage, mitochondrial dysfunction, and increased calpain messenger RNA. Blocking extracellular calcium influx or intracellular calcium release was each effective in suppressing myocyte apoptosis. Cell death was also found to be mediated by calcium sensitive signal transduction events based on the use of specific antagonists. Consistent with the induction of calpain expression during apoptosis, blocking de novo protein synthesis and calpain activity inhibited cell death. These regulatory features coupled with the ease of the cell system suggest that the myocyte apoptosis model described here should be useful in the study of events leading to the demise of the myocardium.

Assessment of a Nuclear Affinity Labeling Method for Tracking Implanted Mesenchymal Stem Cells

Therapeutic implantation of mesenchymal stem cells (MSCs) is entering the realm of clinical trials for several human diseases, and yet much remains uncertain regarding their dynamic distribution and cell fate after in vivo application. Discrepancies in the literature can be attributed in part to the use of different cell labeling/tracking methods and cell administration protocols. To identify a stem cell detection method suitable for myocardial implantation in a large animal model, we experimented on three different MSC labeling methods: adenovirus-mediated expression of enhanced green fluorescence protein (EGFP) and β-galactosidase (LacZ), and nuclear staining with DAPI. Intramuscular and intracoronary administrations of labeled porcine MSCs identified the nuclear affinity dye to be a reliable stem cell tracking marker. Stem cell identification is facilitated by an optimized live cell labeling condition generating bright blue fluorescence sharply confined to the nucleus. DAPI-labeled MSCs retained full viability, ceased proliferation, and exhibited an increased differentiation potential. The labeled MSCs remained fully active in expressing key growth factor and cytokine genes, and notably exhibited enhanced expression of the chemokine receptor CXCR4 and its ligand SDF1, indicating their competency in response to tissue injury. Histological analysis revealed that approximately half a million MSCs or ~2% of the administered MSCs remained localized in the normal pig heart 2 weeks after coronary infusion. That the vast majority of these identified MSCs were interstitial indicated the ability of MSCs to migrate across the coronary endothelium. No evidence was obtained indicating MSC differentiation to cardiomyocyte.