Ji Hyun Choi

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.

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.

Transplantation of Immortalized CD34+ and CD34- Adipose-Derived Stem Cells Improve Cardiac Function and Mitigate Systemic Pro-Inflammatory Responses

Adipose-derived stem cells (ADSCs) have the potential to differentiate into various cell lineages and they are easily obtainable from patients, which makes them a promising candidate for cell therapy. However, a drawback is their limited life span during in vitro culture. Therefore, hTERT-immortalized CD34+ and CD34- mouse ADSC lines (mADSCshTERT) tagged with GFP were established. We evaluated the proliferation capacity, multi-differentiation potential, and secretory profiles of CD34+ and CD34- mADSCshTERT in vitro, as well as their effects on cardiac function and systemic inflammation following transplantation into a rat model of acute myocardial infarction (AMI) to assess whether these cells could be used as a novel cell source for regeneration therapy in the cardiovascular field. CD34+ and CD34- mADSCshTERT demonstrated phenotypic characteristics and multi-differentiation potentials similar to those of primary mADSCs. CD34+ mADSCshTERT exhibited a higher proliferation ability compared to CD34- mADSCshTERT, whereas CD34- mADSCshTERT showed a higher osteogenic differentiation potential compared to CD34+ mADSCshTERT. Primary mADSCs, CD34+, and CD34- mADSCshTERT primarily secreted EGF, TGF-β1, IGF-1, IGF-2, MCP-1, and HGFR. CD34+ mADSCshTERT had higher secretion of VEGF and SDF-1 compared to CD34- mADSCshTERT. IL-6 secretion was severely reduced in both CD34+ and CD34- mADSCshTERT compared to primary mADSCs. Transplantation of CD34+ and CD34- mADSCshTERT significantly improved the left ventricular ejection fraction and reduced infarct size compared to AMI-induced rats after 28 days. At 28 days after transplantation, engraftment of CD34+ and CD34- mADSCshTERT was confirmed by positive Y chromosome staining, and differentiation of CD34+ and CD34- mADSCshTERT into endothelial cells was found in the infarcted myocardium. Significant decreases were observed in circulating IL-6 levels in CD34+ and CD34- mADSCshTERT groups compared to the AMI-induced control group. Transplantation of CD34- mADSCshTERT significantly reduced circulating MCP-1 levels compared to the AMI control and CD34+ mADSCshTERT groups. GFP-tagged CD34+ and CD34- mADSCshTERT are valuable resources for cell differentiation studies in vitro as well as for regeneration therapy in vivo.

Intrinsic FGF2 and FGF5 promotes angiogenesis of human aortic endothelial cells in 3D microfluidic angiogenesis system

The human body contains different endothelial cell types and differences in their angiogenic potential are poorly understood. We compared the functional angiogenic ability of human aortic endothelial cells (HAECs) and human umbilical vein endothelial cells (HUVECs) using a three-dimensional (3D) microfluidic cell culture system. HAECs and HUVECs exhibited similar cellular characteristics in a 2D culture system; however, in the 3D microfluidic angiogenesis system, HAECs exhibited stronger angiogenic potential than HUVECs. Interestingly, the expression level of fibroblast growth factor (FGF)2 and FGF5 under vascular endothelial growth factor (VEGF)-A stimulation was significantly higher in HAECs than in HUVECs. Moreover, small interfering RNA-mediated knockdown of FGF2 and FGF5 more significantly attenuated vascular sprouting induced from HAECs than HUVECs. Our results suggest that HAECs have greater angiogenic potential through FGF2 and FGF5 upregulation and could be a compatible endothelial cell type to achieve robust angiogenesis.

Cardiovascular event rates in patients with ST-elevation myocardial infarction were lower with early increases in mobilization of Oct4highNanoghigh stem cells into the peripheral circulation during a 4-year follow-up

BACKGROUND Long-term clinical implications of embryonic stem cell markers such as Oct4 and Nanog have not been investigated in ST-elevation myocardial infarction (STEMI) patients. The aim of this study was to investigate the effects of early peripheral mobilization of stem cells with Oct4 and Nanog gene expression on major adverse cardiovascular events (MACEs) in patients with STEMI during a 4-year follow-up. METHODS Peripheral blood mononuclear cells (PBMCs) were isolated on days 0, 1 and 7 from patients with STEMI (n = 40) and healthy controls (n=20). The numbers of CD34+, CD117+, CD133+ and c-met+ stem cells were measured by flow-cytometry. Oct4 and Nanog gene expressions were analyzed by real-time PCR. MACEs such as non-fatal MI, death, stroke, target lesion and revascularization were observed. RESULTS MACEs were significantly lower in patients with Oct4 gene expression ≥ 1.13 and Nanog gene expression ≥ 1.20 at admission. The numbers of CD34+, CD117+, CD133+ and c-met+ cells within 7 days after STEMI did not show significant differences in patients with or without MACE. Level of anti-inflammatory marker such as IL-10 was significantly higher within 7 days following STEMI in patients without MACE. Inflammatory and angiogenic markers such as CRP, IL-6, SCF, SDF-1α, and VEGF did not show significant differences in patients with or without MACE. CONCLUSION mRNA levels of pluripotent embryonic stem cell markers such as Oct4 and Nanog were significantly higher in STEMI patients without MACEs during a 4-year follow-up. Baseline Oct4 and Nanog gene expression levels could be used as predictors of MACE in STEMI patients.

Black Raspberry Extract Increased Circulating Endothelial Progenitor Cells and Improved Arterial Stiffness in Patients with Metabolic Syndrome: A Randomized Controlled Trial

Administration of black raspberry (Rubus occidentalis) is known to improve vascular endothelial function in patients at a high risk for cardiovascular (CV) disease. We investigated short-term effects of black raspberry on circulating endothelial progenitor cells (EPCs) and arterial stiffness in patients with metabolic syndrome. Patients with metabolic syndrome (n = 51) were prospectively randomized into the black raspberry group (n = 26, 750 mg/day) and placebo group (n = 25) during the 12-week follow-up. Central blood pressure, augmentation index, and EPCs, such as CD34/KDR(+), CD34/CD117(+), and CD34/CD133(+), were measured at baseline and at 12-week follow-up. Radial augmentation indexes were significantly decreased in the black raspberry group compared to the placebo group (-5% ± 10% vs. 3% ± 14%, P < .05). CD34/CD133(+) cells at 12-week follow-up were significantly higher in the black raspberry group compared to the placebo group (19 ± 109/μL vs. -28 ± 57/μL, P < .05). Decreases from the baseline in interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) were significantly greater in the black raspberry group compared to the placebo group (-0.5 ± 1.4 pg/mL vs. -0.1 ± 1.1 pg/mL, P < .05 and -5.4 ± 4.5 pg/mL vs. -0.8 ± 4.0 pg/mL, P < .05, respectively). Increases from the baseline in adiponectin levels (2.9 ± 2.1 μg/mL vs. -0.2 ± 2.5 μg/mL, P < .05) were significant in the black raspberry group. The use of black raspberry significantly lowered the augmentation index and increased circulating EPCs, thereby improving CV risks in patients with metabolic syndrome during the 12-week follow-up.

Mixl1 and Flk1 Are Key Players of Wnt/TGF-β Signaling During DMSO-Induced Mesodermal Specification in P19 cells

Dimethyl sulfoxide (DMSO) is widely used to induce multilineage differentiation of embryonic and adult progenitor cells. To date, little is known about the mechanisms underlying DMSO‐induced mesodermal specification. In this study, we investigated the signaling pathways and lineage‐determining genes involved in DMSO‐induced mesodermal specification in P19 cells. Wnt/β‐catenin and TGF‐β superfamily signaling pathways such as BMP, TGF‐β and GDF1 signaling were significantly activated during DMSO‐induced mesodermal specification. In contrast, Nodal/Cripto signaling pathway molecules, required for endoderm specification, were severely downregulated. DMSO significantly upregulated the expression of cardiac mesoderm markers but inhibited the expression of endodermal and hematopoietic lineage markers. Among the DMSO‐activated cell lineage markers, the expression of Mixl1 and Flk1 was dramatically upregulated at both the transcript and protein levels, and the populations of Mixl1+, Flk1+ and Mixl1+/Flk1+ cells also increased significantly. DMSO modulated cell cycle molecules and induced cell apoptosis, resulting in significant cell death during EB formation of P19 cells. An inhibitor of Flk1, SU5416 significantly blocked expressions of TGF‐β superfamily members, mesodermal cell lineage markers and cell cycle molecules but it did not affect Wnt molecules. These results demonstrate that Mixl1 and Flk1 play roles as key downstream or interacting effectors of Wnt/TGF‐β signaling pathway during DMSO‐induced mesodermal specification in P19 cells. J. Cell. Physiol. 230: 1807–1821, 2015.

Cyclosporin A Induces Cardiac Differentiation but Inhibits Hemato-Endothelial Differentiation of P19 Cells

Little is known about the mechanisms underlying the effects of Cyclosporin A (CsA) on the fate of stem cells, including cardiomyogenic differentiation. Therefore, we investigated the effects and the molecular mechanisms behind the actions of CsA on cell lineage determination of P19 cells. CsA induced cardiomyocyte-specific differentiation of P19 cells, with the highest efficiency at a concentration of 0.32 μM during embryoid body (EB) formation via activation of the Wnt signaling pathway molecules, Wnt3a, Wnt5a, and Wnt8a, and the cardiac mesoderm markers, Mixl1, Mesp1, and Mesp2. Interestingly, cotreatment of P19 cells with CsA plus dimethyl sulfoxide (DMSO) during EB formation significantly increases cardiac differentiation. In contrast, mRNA expression levels of hematopoietic and endothelial lineage markers, including Flk1 and Er71, were severely reduced in CsA-treated P19 cells. Furthermore, expression of Flk1 protein and the percentage of Flk1+ cells were severely reduced in 0.32 μM CsA-treated P19 cells compared to control cells. CsA significantly modulated mRNA expression levels of the cell cycle molecules, p53 and Cyclins D1, D2, and E2 in P19 cells during EB formation. Moreover, CsA significantly increased cell death and reduced cell number in P19 cells during EB formation. These results demonstrate that CsA induces cardiac differentiation but inhibits hemato-endothelial differentiation via activation of the Wnt signaling pathway, followed by modulation of cell lineage-determining genes in P19 cells during EB formation.

Enhanced cardiomyogenic differentiation of P19 embryonal carcinoma stem cells.

Background and Objectives We investigated the effects of different concentrations of serum, 5-azacytidine, and culture time on the cardiomyogenic differentiation of P19 embryonal carcinoma stem cells in the course of developing an efficient protocol for generating the cardiomyogenic lineage. Materials and Methods P19 cells were plated at a density of 1×106 cells on 10-cm bacterial dishes for 96 hours in the presence of 1% dimethyl sulfoxide to form embryoid bodies. The embryoid bodies were cultured in medium with 2% or 10% fetal bovine serum for an additional 10 or 15 consecutive days in the presence of 0, 1, or 3 µM 5-azacytidine. Results Quantitative real-time polymerase chain reaction (PCR) analysis showed that the messenger ribonucleic acid (mRNA) expression of cardiac muscle-specific genes, such as GATA4, α-actin, α-myosin heavy chain, and cardiac troponin T, were significantly higher in the 15-day culture groups than in the 10-day culture groups. Furthermore, the cardiac muscle-specific genes were expressed more in the high-serum groups compared to the low-serum groups regardless of the culture time. Cardiomyogenic differentiation of the P19 cells was most effective in 1 µM 5-azacytidine regardless of the serum concentrations. In addition, the stimulation effects of 5-azacytidine on cardiomyogenic differentiation were more significant under low-serum culture conditions compared to high-serum culture conditions. Cardiomyogenic differentiation of P19 cells was further confirmed by immunostaining with cardiac muscle-specific antibodies. Conclusion Taken together, these results demonstrated that cardiomyogenic differentiation of P19 cells was enhanced by a combination of different experimental factors.

Cardiac Stem Cell Secretome Protects Cardiomyocytes from Hypoxic Injury Partly via Monocyte Chemotactic Protein-1-Dependent Mechanism

Cardiac stem cells (CSCs) were known to secrete diverse paracrine factors leading to functional improvement and beneficial left ventricular remodeling via activation of the endogenous pro-survival signaling pathway. However, little is known about the paracrine factors secreted by CSCs and their roles in cardiomyocyte survival during hypoxic condition mimicking the post-myocardial infarction environment. We established Sca-1+/CD31− human telomerase reverse transcriptase-immortalized CSCs (Sca-1+/CD31− CSCshTERT), evaluated their stem cell properties, and paracrine potential in cardiomyocyte survival during hypoxia-induced injury. Sca-1+/CD31− CSCshTERT sustained proliferation ability even after long-term culture exceeding 100 population doublings, and represented multi-differentiation potential into cardiomyogenic, endothelial, adipogenic, and osteogenic lineages. Dominant factors secreted from Sca-1+/CD31− CSCshTERT were EGF, TGF-β1, IGF-1, IGF-2, MCP-1, HGF R, and IL-6. Among these, MCP-1 was the most predominant factor in Sca-1+/CD31− CSCshTERT conditioned medium (CM). Sca-1+/CD31− CSCshTERT CM increased survival and reduced apoptosis of HL-1 cardiomyocytes during hypoxic injury. MCP-1 silencing in Sca-1+/CD31− CSCshTERT CM resulted in a significant reduction in cardiomyocyte apoptosis. We demonstrated that Sca-1+/CD31− CSCshTERT exhibited long-term proliferation capacity and multi-differentiation potential. Sca-1+/CD31− CSCshTERT CM protected cardiomyocytes from hypoxic injury partly via MCP-1-dependent mechanism. Thus, they are valuable sources for in vitro and in vivo studies in the cardiovascular field.