Abstract 51: Nucleostemin Induced by Pim-1 Kinase Is Critical to Maintain Pluripotency and Enhance Regenerative Potential of Cardiac Progenitor Cells

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Nirmala Hariharan ◽  
Anya Y Joyo ◽  
Kaitlen M Samse ◽  
Daniele Avitabile ◽  
Brandi Bailey ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Shape Control ◽  
Kinase Inhibitor ◽  
Cardiac Regeneration ◽  
Stem Cell Marker ◽  
Cardiac Progenitor Cells ◽  
Protein Levels ◽  
Regulation Of Cell Cycle ◽  
Factor C

Myocardial regeneration and repair in response to injury are governed in part by cell survival, proliferation and pluripotency. Proliferation and survival in cardiac progenitor cells (CPCs) are mediated by Pim-1, a serine threonine kinase, and nucleostemin (NS), a nucleolar stress sensor protein. The role of NS in regulating CPC pluripotency and the molecular mechanism of NS induction and action is largely unknown. The hypothesis of the study is that NS, induced by Pim-1 mediated stabilization of transcription factor c-Myc is critical to maintain CPC pluripotency and inhibits senescence. NS and c-Myc protein levels are increased in cultured CPCs overexpressing Pim-1 (3.1 and 5.5 fold, p<0.01) while knockdown of Pim-1 using sh-RNA decreases c-Myc and NS expression (-60%, -54%, p<0.05), similar to effects mediated by a Pim-1 kinase inhibitor (p<0.01), indicating that Pim-1 regulates both c-Myc and NS. c-Myc is necessary and sufficient for NS regulation, as indicated by the increase (3.1 fold, p<0.01) and decrease (-60.2%, p<0.01) in NS expression upon lentiviral mediated over-expression or knockdown of c-Myc, respectively. Regulation of NS promoter by c-Myc is evident from loss of GFP expression and fluorescence following knock down of c-Myc in CPCs isolated from transgenic mice expressing eGFP driven by the NS promoter. The role of NS in regulating CPC pluripotency is determined by silencing NS. Change in morphology (flat, round cells vs spindle shape control CPCs), decreased expression of stem cell marker c-Kit (-55%, p<0.05), up-regulation of cell cycle inhibitors p53 and p16 (4.2, 3.8 fold, p<0.01) and decreased proliferation (p<0.05) result from loss of NS in CPCs, suggestive of increased senescence and loss of pluripotency. NS-mediated regulation of CPC senescence is p53 dependent, as silencing p53 reverses CPC morphology and pluripotency lost by NS depletion. In conclusion, NS which is induced downstream of Pim-1 kinase maintains pluripotency and enhances regenerative potential in CPCs. These findings are consistent with cumulative evidence that Pim-1 induced cardiac regeneration is mediated in part by NS, providing an additional mechanistic basis for benefits of genetic engineering with Pim-1 to enhance NS expression in cardiac stem cells.

Abstract 1839: MicroRNA-499 Promotes the Differentiation of Cardiac Progenitor Cells into Myocytes

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Toru Hosoda ◽  
Konrad Urbanek ◽  
Adriana Bastos Carvalho ◽  
Claudia Bearzi ◽  
Silvana Bardelli ◽  
...  
Keyword(s):  
Gap Junctions ◽  
Progenitor Cells ◽  
Target Genes ◽  
Brdu Incorporation ◽  
Cardiac Progenitor Cells ◽  
Partial Recovery ◽  
Rt Pcr ◽  
Protein Levels ◽  
Cell Proliferation And Differentiation ◽  
Reporter Assays

Myocardial regeneration mediated by cardiac progenitor cells (CPCs) results in the partial recovery of the infarcted heart but the newly formed myocytes within the necrotic tissue have fetal-neonatal characteristics. In contrast, CPC activation in the remote viable myocardium results in the formation of mature myocytes, suggesting that CPC differentiation is conditioned by the surrounding cells. Thus, the hypothesis is raised that microRNAs (miRs) that are highly expressed in myocytes and are absent in CPCs, may translocate through gap junctions to adjacent CPCs promoting their differentiation. By employing miR array and Q-RT-PCR, miR-499 was found to be ~500-fold more expressed in myocytes than CPCs. Additionally, we demonstrated that miR-499 translocates from neighboring cells to CPCs through the formation of gap junctions. The translocated miR-499 was functional and repressed the expression of target genes. Among 200 putative targets of miR-499, we have elected to study Sox6 and Rod1. The validation of these putative miR-499-targets was obtained by reporter assays; cells transfected with miR-499 together with plasmids carrying luciferase and the 3′-UTR region of Sox6 or Rod1 show the expected decrease in luciferase activity. Transcripts of Sox6 and Rod1 were measured by Q-RT-PCR in myocytes and CPCs; Sox6 mRNA was 2-fold higher and Rod1 mRNA was 98% lower in myocytes than CPCs. However, the protein levels of Sox6 and Rod1 were significantly lower in myocytes than CPCs suggesting that miR-499 promotes degradation and/or inhibition of translation of these target genes. To document miR-499 function, CPCs were transfected with a miR-499-expression vector and cell proliferation and differentiation were evaluated 3 days later. BrdU incorporation decreased 60% and the cells displayed a marked upregulation of the myocyte-specific transcription factors Nkx2.5 and MEF2C. Similar results were obtained when Sox6 and Rod1 were selectively blocked with siRNA. In both cases, the number of Nkx2.5- and MEF2C-positive cells increased 2–3-fold. Thus, our data indicate that miR-499 translocates via gap junction from myocytes to CPCs where miR-499 is a crucial modulator of the differentiation of CPCs into cardiomyocytes through the repression of Sox6 and Rod1.

Abstract 18698: Identification of a Novel Cardiac Progenitor Population Expressing Pw1/peg3 in the Murine Heart

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Elisa Yaniz-Galende ◽  
Luigi Formicola ◽  
Nathalie Mougenot ◽  
Lise Legrand ◽  
Jiqiu Chen ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Fold Increase ◽  
Cardiac Regeneration ◽  
Cardiac Repair ◽  
Cardiac Progenitor Cells ◽  
Average Percentage ◽  
Pathological Conditions ◽  
Reporter Mice ◽  
Novel Target

The myocardium responds to injury by recruiting cardiac progenitor cells (CPCs) to the injured tissue to promote cardiac repair. Although different classes of CPCs have been identified, their contribution in physiological and pathological conditions remains unclear. PW1 gene has recently been proposed as a marker of resident adult stem and progenitor cell populations in several adult tissues. Our goal was to characterize and determine the role of PW1+ population in the heart. Here, we employ immunostaining and fluorescence-activated cell sorting (FACS) analysis in PW1-reporter mouse to perform qualitative and quantitative analyses of PW1+ population in the heart. We first found that PW1+ cells are mainly located in the epicardium and myocardial interstitium of normal hearts. The average percentage of PW1+ cells, as assessed by FACS, was 1.56±1.41%. A subset of PW1+ cells also co-express other CPC markers such as Sca-1 (52±22%) or PDGFR1α (43±14%). In contrast, a very small proportion of PW1+ cells co-express c-kit (6±5%). To investigate the contribution of PW1+ cells in pathological conditions, we then performed myocardial infarction (MI) by LAD ligation in PW1-reporter mice. We found that MI resulted in a 3-fold increase in the number of PW1+ cells in infarcted mice compared with sham-operated groups, at 1 week post-MI (1.16%±0.47% in sham versus 3.43%±0.82 in MI). This population preferentially localized in the injured myocardium and border area. PW1+ cells were isolated by FACS from the whole infarcted heart from PW1-reporter mice. In vitro differentiation assays reveal that purified PW1+ cells are multipotent and can spontaneously differentiate into smooth muscle cells, endothelial cells and cardiomyocyte-like cells. Taken together, our data identify a novel PW1+ cardiac progenitor population with the potential to undergo differentiation into multiple cardiac lineages, suggesting their involvement in cardiac repair in normal and pathological conditions. The discovery of a novel population of cardiac progenitor cells, augmented following MI and with cardiogenic potential, provides a novel target for therapeutic approaches aimed at improving cardiac regeneration.

scholarly journals Phenotypic Screen with the Human Secretome Identifies FGF16 as Inducing Proliferation of iPSC-Derived Cardiac Progenitor Cells

2019 ◽  
Vol 20 (23) ◽  
pp. 6037 ◽  
Author(s):  
Karin Jennbacken ◽  
Fredrik Wågberg ◽  
Ulla Karlsson ◽  
Jerry Eriksson ◽  
Lisa Magnusson ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cardiac Fibroblasts ◽  
Cardiac Regeneration ◽  
Cardiac Cells ◽  
Mouse Heart ◽  
Cardiac Progenitor Cells ◽  
High Sequence Identity ◽  
Paracrine Factors ◽  
Human Cardiomyocytes ◽  
Phenotypic Screen

Paracrine factors can induce cardiac regeneration and repair post myocardial infarction by stimulating proliferation of cardiac cells and inducing the anti-fibrotic, antiapoptotic, and immunomodulatory effects of angiogenesis. Here, we screened a human secretome library, consisting of 923 growth factors, cytokines, and proteins with unknown function, in a phenotypic screen with human cardiac progenitor cells. The primary readout in the screen was proliferation measured by nuclear count. From this screen, we identified FGF1, FGF4, FGF9, FGF16, FGF18, and seven additional proteins that induce proliferation of cardiac progenitor cells. FGF9 and FGF16 belong to the same FGF subfamily, share high sequence identity, and are described to have similar receptor preferences. Interestingly, FGF16 was shown to be specific for proliferation of cardiac progenitor cells, whereas FGF9 also proliferated human cardiac fibroblasts. Biosensor analysis of receptor preferences and quantification of receptor abundances suggested that FGF16 and FGF9 bind to different FGF receptors on the cardiac progenitor cells and cardiac fibroblasts. FGF16 also proliferated naïve cardiac progenitor cells isolated from mouse heart and human cardiomyocytes derived from induced pluripotent cells. Taken together, the data suggest that FGF16 could be a suitable paracrine factor to induce cardiac regeneration and repair.

Role of SDF-1Α/CXCR4 axis in the homing and uptake of cardiac progenitor cells derived exosomes by damaged cardiomyocytes

2018 ◽  
Vol 103-105 ◽  
pp. 52
Author(s):  
A. Ciullo ◽  
V. Biemmi ◽  
G. Milano ◽  
E. Cervio ◽  
S. Bolis ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Cardiac Progenitor Cells ◽  
Cxcr4 Axis

scholarly journals Gremlin2 Regulates the Differentiation and Function of Cardiac Progenitor Cells via the Notch Signaling Pathway

2018 ◽  
Vol 47 (2) ◽  
pp. 579-589 ◽  
Author(s):  
Wei Li ◽  
Yaojun Lu ◽  
Ruijuan Han ◽  
Qiang Yue ◽  
Xiurong Song ◽  
...  
Keyword(s):  
Mouse Model ◽  
Progenitor Cells ◽  
Left Ventricular ◽  
Cardiac Repair ◽  
Cardiac Progenitor Cells ◽  
Cardiomyocyte Differentiation ◽  
Rt Pcr ◽  
And Function

Background/Aims: The transplantation of cardiac progenitor cells (CPCs) improves neovascularization and left ventricular function after myocardial infarction (MI). The bone morphogenetic protein antagonist Gremlin 2 (Grem2) is required for early cardiac development and cardiomyocyte differentiation. The present study examined the role of Grem2 in CPC differentiation and cardiac repair. Methods: To determine the role of Grem 2 during CPC differentiation, c-Kit+ CPCs were cultured in differentiation medium for different times, and Grem2, Notch1 and Jagged1 expression was determined by RT-PCR and western blotting. Short hairpin RNA was used to silence Grem2 expression, and the expression of cardiomyocyte surface markers was assessed by RT-PCR and immunofluorescence staining. In vivo experiments were performed in a mouse model of left anterior descending coronary artery ligation-induced MI. Results: CPC differentiation upregulated Grem2 expression and activated the Notch1 pathway. Grem2 knockdown inhibited cardiomyocyte differentiation, and this effect was similar to that of Notch1 pathway inhibition in vitro. Jagged1 overexpression rescued the effects of Grem2 silencing. In vivo, Grem2 silencing abolished the protective effects of CPC injection on cardiac fibrosis and function. Conclusions: Grem2 regulates CPC cardiac differentiation by modulating Notch1 signaling. Grem2 enhances the protective effect of CPCs on heart function in a mouse model of MI, suggesting its potential as the rapeutic protein for cardiac repair.

Abstract 4: Lymphatic Vessels Mediate the Mobilization of Cardiac Progenitor Cells after Myocardial Infarction

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Polina Goichberg ◽  
Maria Cimini ◽  
Antonio Cannata ◽  
Sergio Signore ◽  
Kanako Waight ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Progenitor Cells ◽  
Heart Diseases ◽  
Lymphatic Vessels ◽  
Mouse Heart ◽  
Cardiac Progenitor Cells ◽  
Myocardial Repair ◽  
Lymphatic Circulation ◽  
Lymphatic Vasculature

The delivery of adult cardiac progenitor cells (CPCs) or their activation in situ constitute an evolving approach for the treatment of heart failure. CPCs are endowed with regenerative capacity, producing differentiating myocytes and vascular structures in the course of homeostasis and upon injury. The regenerative function of CPCs is contingent to their ability to migrate to and engraft within the wounded area. Yet, the mechanisms governing CPC trafficking in the diseased myocardium are largely unknown. The lymphatic system is vital for tissue repair, and the role of the lymphatic vasculature in the trafficking of hematopoietic and cancer cells is well documented. We examined whether cardiac lymphatic vessels mediate the translocation of CPCs in the infarcted myocardium. By imaging of the heart from transgenic c-kit-GFP reporter mice, we found that as early as 4 hours after myocardial infarction (MI), uncommitted lineage-negative progenitors accumulated in the vicinity of the lymphatic vessels located in the region bordering the necrotic area. Histologically, extensive lymphangiogenesis was documented in the mouse heart in the acute (8-48 hours) and chronic (15-35 days) phases of infarct healing and scar formation. CPCs were detected traversing the wall of lymphatic vessels at different stages after MI, indicative of the functional role of the lymphatic circulation in the recruitment of primitive cells to the site of injury. Furthermore, isolated human CPCs exhibited chemotaxis and specific binding to the human lymphatic endothelial cells (LECs) in steady-state conditions and, increasingly, after exposure to an inflammatory cytokine, TNFα. CPCs performed trans-endothelial migration in vitro, and actively intravasated into the lumen of microvessels formed by LECs in three-dimensional matrices. Finally, our data suggest that sphingosine-1-phosphate (S1P)-stimulated signaling governs the interactions of CPCs with LECs. These findings on the direct role of lymphatic vasculature in CPC trafficking may contribute to the development of novel therapeutic modalities to increase mobilization of endogenous or transplanted CPCs, promoting myocardial repair in patients with ischemic heart diseases.

HOXA10 Expression Induces by Abl Kinase Inhibitors Enhanced Apoptosis through PI3K Pathway in CML Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4529-4529
Author(s):  
Isao Hirano ◽  
Yuya Sugimoto ◽  
Keiji Okinaka ◽  
Takaaki Ono ◽  
Kaori Shinjo ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Pi3k Pathway ◽  
Hematopoietic Progenitor ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Abl Kinase ◽  
Abl Kinase Inhibitors

Abstract [Background] Homeobox (Hox) genes are grouped together in 4 clusters, A to D. Recent studies has shown that the Hox proteins are important in the control of differentiation and proliferation in hematopoietic cells. We found that the Abl kinase inhibitors increased the expression of HoxA10 gene in CML cells. In this study, we analyzed the role of HoxA10 in CML cell lines and the hematopoietic progenitor cells derived from CML patients by inhibiting the expression of HoxA10. Moreover, we investigated whether the regulation of HoxA10 eradicate Ph+ hematopoietic stem/progenitor cells, which are the targets for leukemic transformation in CML. [Methods] We used AMN107 and BMS354825 for the Abl kinase inhibitors, LY294002 for a PI3K inhibitor, PP2 for a Src kinase inhibitor, and SB203580 for a p38 MAP kinase inhibitor. For analysis of HoxA10 mRNA and protein, RT-PCR and western blot were performed in K562, Meg-01 and U937 cells, which untreated or treated with AMN107, BMS354825, LY294002, PP2, or SB203580 respectively. We then attempted to localize the intracellular locations of HoxA10 in K562 and Meg01, which untreated or treated with AMN107, BMS354825, or LY294002 by using conforcal fluorescence microscopy. For analysis of proliferation in K562, Meg-01 and U937 transfected with siRNA HoxA10, MTT assays were performed in untransfected or transfected K562, Meg-01 and U937 treated with or without AMN107, BMS354825, or LY294002. Finally, we counted the colony numbers of CFU-GEMM, CFU-GM, and BFU-E in K562 and Meg-01 treated with the Abl kinase inhibitors or LY294002. Results Both K562 and Meg01 cells expressed HoxA10 mRNA and protein at lower level compared to U937 cells. Interestingly, treatment with AMN107, BMS354825, or LY294002 increased the expression of HoxA10 mRNA and protein in both K562 and Meg01 cells. The fluorescence of HoxA10 was more strongly observed in the area corresponding to the cell’s cytoplasm than nucleus, and the treatment with AMN107, BMS354825, or LY294002 increased the fluorescence in nucleus of K562 and Meg01 cells in a time-dependent manner. In K562 and Meg01 cells transfected with the siRNA HoxA10, treatment with AMN107 or BMS354825 slightly inhibited the proliferation compared to K562 and Meg01 transfected with control siRNA. Finally, we showed that the inhibition of HoxA10 expression by siRNA increased the numbers of CFU-GEMM, BFU-E, and CFU-GM when the cells were treated with the combination of BMS354825 and LY294002 compared to control cells. [Conclusions] In this study, we showed that the Abl kinase inhibitors induced the expression of HoxA10, and HoxA10 was regulated by PI3K pathway in CML cells. This finding indicates a new insight in the regulation of cell proliferation via the PI3K signal pathway in CML cells. Moreover, we found the role of HoxA10 in CML cell lines and the hematopoietic progenitor cells derived from CML patients by inhibiting the expression of HoxA10. We showed that the regulation of HoxA10 eradicated Bcr-Abl+ hematopoietic stem/progenitor cells, which are the targets for leukemic transformation in CML.

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