Seung Cheol Choi

5-azacytidine induces cardiac differentiation of P19 embryonic stem cells.

The P19 embryonal carcinoma cell line is a useful model cells for studies on cardiac differentiation. However, its low efficacy of differentiation hampers its usefulness. We investigated the effect of 5-azacytidine (5-aza) on P19 cells to differentiate into a high-efficacy cardiomyocytes. Embryoid-body-like structures were formed after 6 days with 1 µM of 5-aza in a P19 cell monolayer culture, beating cell clusters first observed on day 12, and, the production of beating cell clusters increased by 80.1% (29 of 36-wells) after 18 days. In comparison, the spontaneous beating cells was 33.3% (12 of 36-wells) for the untreated control cells. In response to 1 µM of 5-aza, P19 cells expressed bone morphogenetic protein-2 (BMP-2), BMP-4, Bmpr1a and Smad1 at day 6 or 9, and also cardiac markers such as GATA-4, Nkx2.5, cardiac troponin I, and desmin were up-regulated in a time-dependent manner after induction of BMP signaling molecules. Immunocytochemistry revealed the expression of smooth muscle a-actin, sarcomeric a-actinin, cardiac myosin heavy chain, cardiac troponin T and desmin, respectively. The proportion of sarcomeric a-actinin positive cells accounted for 6.48% on day 15 after 5-aza exposure as measured by flow cytometry. This study has demonstrated that 5-aza induces differentiation of P19 cells into cardiomyocytes in a confluent monolayer culture in the absence of prior embryoid formation and dimethyl sulfoxide exposure, depending in part on alteration of BMP signaling molecules. These results suggest that 5-aza treatment could be used as a new method for cardiac differentiation in P19 cells.

Fibroblast growth factor-2 and -4 promote the proliferation of bone marrow mesenchymal stem cells by the activation of the PI3K-Akt and ERK1/2 signaling pathways.

Bone marrow mesenchymal stem cells (BMMSCs) have the capacity for self-renewal, and differentiation into a variety of cell types. They thus represent an attractive source of material for cell therapy. However, little is known about the mechanisms underlying the proliferation of BMMSCs. The purpose of this study was to identify the factors and signaling pathways involved in the proliferation of stem cell antigen-1(+) (Sca-1(+)) BMMSCs. Among the cytokines and growth factors examined in this study, fibroblast growth factor-2 (FGF-2) and FGF-4 significantly stimulated the proliferation of Sca-1(+) BMMSCs, as determined by bromodeoxyuridine incorporation. PI3K-Akt, ERK1/2, and JAK/STAT3 pathways were investigated after stimulation with FGF-2 or FGF-4 via Western blot analysis. No changes were observed in the total ERK1/2 and Akt; however, the pERK1/2 and pAkt levels were upregulated early within 15 min in the FGF-2- or FGF-4-treated Sca-1(+) BMMSCs. Moreover, the pERK1/2 and pAkt upregulation induced by FGF-2 and -4 were completely abolished by treatment with the MEK1/2 inhibitor, U0126 and the PI3K inhibitor, LY294002. However, no change in pJAK2 or total JAK2 levels was observed in the Sca-1(+) BMMSCs induced by FGF-2 or FGF-4. As a consequence of PI3K-Akt and ERK1/2, the upregulation of c-Jun in the Sca-1(+) BMMSCs, after stimulation with FGF-2 or FGF-4, was observed after 12 and 24 h. Moreover, the activation of c-Jun in FGF-2- and FGF-4-treated Sca-1(+) BMMSCs was significantly reduced by U0126. Taken together, these data suggest that FGF-2 and -4 promote the proliferation of Sca-1(+) BMMSCs by activation of the ERK1/2 and PI3K-Akt signaling pathways.

Isolation and Characterization of Multipotent Human Keloid-Derived Mesenchymal-Like Stem Cells

In this study, we report the isolation and characterization of a population of multipotent keloid-derived mesenchymal-like stem cells (KMLSCs) from keloid scalp tissues. These KMLSCs expressed the typical mesenchymal stem cell marker proteins CD13, CD29, CD44, CD90, fibronectin, and vimentin when they were cultured in serum-containing medium and when subsequent exposure to various differentiation media resulted in their differentiation into adipocytes, osteoblasts, chondrocytes, smooth muscle cells, and angiogenic endothelial cells. When KMLSCs were cultured in neural stem culture conditions (i.e., in the presence of epidermal growth factor and fibroblast growth factor 2 in substrate-free conditions), they produced large numbers of neurospheres containing nestin-, CD133-, and SOX2-positive cells that expressed neural-crest stem cell markers. Subsequent exposure of these cells to different differentiation conditions resulted in cells that expressed neuronal cell-, astrocyte-, oligodendrocyte-, or Schwann cell-specific markers. Our study suggests that KMLSCs may be an alternative adult stem cell resource for regenerative tissue repair and auto-transplantation.

Nanog regulates molecules involved in stemness and cell cycle-signaling pathway for maintenance of pluripotency of P19 embryonal carcinoma stem cells

To identify potential downstream targets of Nanog, a key transcription factor in the maintenance of pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells, global gene expression profiles in Nanog small interfering RNA (siRNA)‐transfected P19 EC stem cells were performed using cDNA, 60‐mer, and 30‐mer microarray platforms. The putative Nanog target genes identified by Nanog silencing were verified using reverse transcription‐polymerase chain reaction after Nanog overexpression. Downregulation of Nanog in P19 cells resulted in reduction of pluripotency markers, such as Fgf4, Klf2, Mtf2, Oct‐4, Rex1, Sox1, Yes, and Zfp143, whereas overexpression of Nanog in P19 cells reversely upregulated their expression. However, expressions of pluripotency markers Cripto, germ cell nuclear factor, Sox2, and Zfp57 as well as leukemia inhibitory factor (LIF)/Stat3 pathway molecules LIF, IL6st, and Stat3 were not affected after 48 h transfection with Nanog siRNA or construct. Nanog silencing also downregulated expression of molecules involved in the p53‐ and cell cycle‐signaling pathway (Atf3, Jdp2, Cul3, Hist1hic, and Bcl6), whereas expression of E2f1, Tob1, Lyn, and Smarcc1 was upregulated by Nanog silencing. Expressions of cyclins D1, D2, D3, and E1 as well as cyclin‐dependent kinase (Cdk) 1 and Cdk6 were downregulated by Nanog silencing in P19 cells, whereas Nanog overexpression reversely increased their expressions. Taken together, examination of global transcriptional changes after Nanog silencing followed by verification by Nanog overexpression has revealed new molecules involved in the maintenance of self‐renewal and in the regulation of the p53‐ and cell cycle‐pathway of P19 cells. J. Cell. Physiol. 227: 3678–3692, 2012.

Telmisartan reduces neointima volume and pulse wave velocity 8 months after zotarolimus-eluting stent implantation in hypertensive type 2 diabetic patients

Objective Telmisartan is a peroxisome proliferator-activated receptor-γ activator with potent anti-inflammatory and antiatherogenic effects. The authors compared the effects of telmisartan and valsartan on neointima volume, atherosclerosis progression and brachial-ankle pulse wave velocity (baPWV) after stenting in hypertensive type 2 diabetes. Design This was a prospective, randomised, 8-month follow-up study that included patients with significant coronary stenosis who received telmisartan (n=36) or valsartan (n=37). Setting University hospital. Main outcome measures Neointima volume and atherosclerosis progression 10 mm proximal and distal to the stented segment were analysed using repeat intravascular ultrasonography. baPWV and inflammatory markers such as interleukin 6, tumour necrosis factor α, C-reactive protein and adiponectin were compared. Results Neointima volume at 8 months was significantly lower in the telmisartan group than the valsartan group (1.9±1.0 vs 2.6±1.4 mm3/1 mm, p=0.007, respectively). Total plaque volumes 10 mm proximal (7.1±1.5 vs 7.8±1.6 mm3/1 mm, p=0.032, respectively) and distal (3.5±1.4 vs 4.1±1.3 mm3/1 mm, p=0.028, respectively) to the stent were significantly lower in the telmisartan group than the valsartan group at 8 months. The decrease from baseline in baPWV was significantly greater in the telmisartan group than the valsartan group (−52±104 vs 30±113 cm/s, p=0.002, respectively). The increase from baseline in adiponectin levels and the decreases from baseline in interleukin 6 and tumour necrosis factor α levels were significantly greater in the telmisartan group at 8 months. Retinol-binding protein-4, homeostasis model of assessment index, hemoglobin A1c and low-density lipoprotein cholesterol levels decreased significantly in both groups without differences in changes from baseline between the two groups. Conclusions Telmisartan reduced neointima volume; atherosclerosis progression 10 mm proximal and distal to the stented segment and baPWV independent of blood pressure, glucose and lipid control in hypertensive type 2 diabetes. Clinical trial no NCT00599885 (clinicaltrials.gov.)

Cardiac side population cells exhibit endothelial differentiation potential

Recent studies have shown that side population (SP) cells, isolated from adult myocardium, represent a distinct cardiac progenitor cell population that exhibits functional cardiomyogenic differentiation. However, information on the intrinsic characteristics and endothelial potential, of cardiac SP cells, is limited. The present study was designed to investigate whether cardiac SP cells exhibit endothelial differentiation potential. The cardiac SP cells more highly expressed the early cardiac transcription factors as well as endothelial cell markers compared to the bone marrow-SP cells. After treatment with VEGF, for 28 days, cardiac SP cells were able to differentiate into endothelial cells expressing von Willebrand factor as determined by immunocytochemistry. Furthermore, expression of endothelial cell markers increased several-fold in VEGF-treated cardiac SP cells compared to the control group when assessed by real-time PCR. We also confirmed that cardiac SP cells provided a significantly augmented ratio of ischemic/normal blood flow, in the cardiac SP cell-transplanted group compared with saline-treated controls on postoperative days 7, 14, 21 and 28, in a murine model. These results show that cardiac SP cells may contribute to regeneration of injured heart tissues partly by transdifferentiation into angiogenic lineages.

Dominant Role of Peroxiredoxin/JNK Axis in Stemness Regulation during Neurogenesis from Embryonic Stem Cells

Redox balance has been suggested as an important determinant of “stemness” in embryonic stem cells (ESCs). In this study, we demonstrate that peroxiredoxin (Prx) plays a pivotal role in maintenance of ESC stemness during neurogenesis through suppression of reactive oxygen species (ROS)‐sensitive signaling. During neurogenesis, Prx I and Oct4 are expressed in a mutually dependent manner and their expression is abruptly downregulated by an excess of ROS. Thus, in Prx I−/− or Prx II−/− ESCs, rapid loss of stemness can occur due to spontaneous ROS overload, leading to their active commitment into neurons; however, stemness is restored by the addition of an antioxidant or an inhibitor of c‐Jun N‐terminal kinase (JNK). In addition, Prx I and Prx II appear to have a tight association with the mechanism underlying the protection of ESC stemness in developing teratomas. These results suggest that Prx functions as a protector of ESC stemness by opposing ROS/JNK cascades during neurogenesis. Therefore, our findings have important implications for understanding of maintenance of ESC stemness through involvement of antioxidant enzymes and may lead to development of an alternative stem cell‐based therapeutic strategy for production of high‐quality neurons in large quantity. Stem Cells 2014;32:998–1011

Red ginseng extract improves coronary flow reserve and increases absolute numbers of various circulating angiogenic cells in patients with first ST-segment elevation acute myocardial infarction.

The effects of red ginseng extract on circulating angiogenic cell mobilization and improvement of microvascular integrity were evaluated in ST‐elevation acute myocardial infarction (AMI) patients during 8‐month follow‐up. AMI patients (n = 50) were randomly assigned to the red ginseng group (3 g/day, n = 25) or the placebo group (n = 25) after coronary stenting. Coronary flow reserve (CFR) was measured at baseline and at 8 months with an intracoronary Doppler wire. Serial changes in the absolute numbers of circulating angiogenic cells such as CD34+, CXCR4+, CD117+, CD133+ and C‐met+ were measured at baseline, 1 day, 5 days and at 8 months.

Intramyocardial transplantation of human adipose-derived stromal cell and endothelial progenitor cell mixture was not superior to individual cell type transplantation in improving left ventricular function in rats with myocardial infarction

BACKGROUND Both adipose-derived stromal cells (ASCs) and endothelial progenitor cells (EPCs) have high potential for promoting tissue revascularization and functional recovery in acute myocardial infarction (AMI) models. We investigated the functional effects of intramyocardial transplantation of a human ASC and EPC mixture in immunodeficient rats after MI. METHODS MI was induced by ligating left anterior descending coronary artery. Survived rats were randomly assigned to 1 of 4 different groups: the control group (n=10, saline in 100μL), the ASC group (n=10, 10(6) ASCs), the EPC group (n=10, 10(6) EPCs), or the ASC+EPC group (n=10, 2×10(5) ASCs+8×10(5) EPCs). Left ventricular (LV) function was compared using echocardiography during the 28-day follow-up. GAP43+ nerve sprouting and smooth muscle α-actin+angiogenesis were also compared. RESULTS Serial changes in LV ejection fraction (EF) and fractional shortening revealed significant increases in the ASC, EPC, and ASC+EPC groups when compared to the control group during the follow-up (49±3%, 49±4%, 47±4%, 39±2%, P<0.001, respectively for LVEF) (33±4%, 32±2%, 31±2%, 23±2%, P=0.002, respectively for fractional shortening). The number of α-actin+arterioles and GAP43+ nerve area was significantly greater in the ASC, EPC, and ASC+EPC groups when compared to the control group in the peri-infarct area (34.4±1.0/mm(2), 35.9±1.1/mm(2), 35.3±0.9/mm(2), 17.4±0.7/mm(2), P<0.001, respectively for angiogenesis) (346.2±10.7μm(2)/mm(2), 357.2±12.8μm(2)/mm(2), 368.0±9.7μm(2)/mm(2), 174.6±7.9μm(2)/mm(2), P<0.001, respectively for nerve sprouting). CONCLUSIONS [corrected] Intramyocardial injections of ASCs, EPCs, or ASCs+EPCs are effective modalities for the treatment of myocardial damage in rats and may expand the potential clinical application of ASC or EPC therapy in patients with ischemic heart disease.

Low-dose versus moderate-dose atorvastatin after acute myocardial infarction: 8-month effects on coronary flow reserve and angiogenic cell mobilisation

Objective To compare the effects of atorvastatin 10 mg versus 40 mg in circulating angiogenic cell mobilisations and in restoring coronary flow reserve (CFR) during the 8-month follow-up in patients with a first acute myocardial infarction (AMI). Design CFR was measured using an intracoronary Doppler wire in 102 patients with AMI at baseline and at 8 months. Changes in the absolute number of circulating angiogenic cells were measured at baseline, 1 day, 5 days and at 8 months. Stented patients were randomly assigned to either low-dose atorvastatin 10 mg (ATOR10, n=52) or moderate-dose atorvastatin 40 mg (ATOR40, n=50). Setting University Hospital. Results CFR increased significantly in both groups during the 8-month follow-up. The 8-month increases from baseline in CFR were significantly greater in the ATOR40 group than in the ATOR10 group (0.99±0.69 vs 0.55±0.47, p=0.017, respectively). The serial increases in the absolute number of CD34+ and CXCR4+ cells were significantly greater in the ATOR40 group, especially at 24 h after the procedure (two-way repeated-measures analysis of variance: p=0.046 and p=0.022, respectively). Decreases from baseline for interleukin 6 (−2.94±3.31 vs −1.52±2.82 pg/ml), tumour necrosis factor α (−1.31±2.96 vs −0.01±1.29 pg/ml), soluble intercellular adhesion molecule-1 (−71±95 vs 37±83 ng/ml) and soluble vascular cell adhesion molecule-1 (−51±364 vs 190±204 ng/ml) were significantly greater in the ATOR40 group. Conclusions The recovery of microvascular integrity after acute ischaemic injury in the ATOR40 group was expedited by greater circulating angiogenic cell mobilisations such as CD34+ and CXCR4+ cells, together with greater decreases in inflammatory cytokines and low-density lipoprotein-cholesterol concentrations. Registration number http://ClinicalTrials.gov number, NCT00536887.