Fabrication of a Nanofibrous Scaffold for the In Vitro Culture of Cardiac Progenitor Cells for Myocardial Regeneration

2013 ◽  
Vol 63 (5) ◽  
pp. 229-239 ◽  
Author(s):  
Rouhollah Mehdinavaz Aghdam ◽  
Saeed Shakhesi ◽  
Siamak Najarian ◽  
Mona Malek Mohammadi ◽  
Seyed Hossein Ahmadi Tafti ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Nanofibrous Scaffold ◽  
Myocardial Regeneration ◽  
Cardiac Progenitor Cells

scholarly journals Novel Identified Circular Transcript of RCAN2, circ-RCAN2, Shows Deviated Expression Pattern in Pig Reperfused Infarcted Myocardium and Hypoxic Porcine Cardiac Progenitor Cells In Vitro

2021 ◽  
Vol 22 (3) ◽  
pp. 1390
Author(s):  
Julia Mester-Tonczar ◽  
Patrick Einzinger ◽  
Johannes Winkler ◽  
Nina Kastner ◽  
Andreas Spannbauer ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Progenitor Cells ◽  
Regulatory Networks ◽  
Circular Rnas ◽  
Cardiac Progenitor Cells ◽  
Tissue Samples ◽  
Healthy Myocardium ◽  
Acute Mi

Circular RNAs (circRNAs) are crucial in gene regulatory networks and disease development, yet circRNA expression in myocardial infarction (MI) is poorly understood. Here, we harvested myocardium samples from domestic pigs 3 days after closed-chest reperfused MI or sham surgery. Cardiac circRNAs were identified by RNA-sequencing of rRNA-depleted RNA from infarcted and healthy myocardium tissue samples. Bioinformatics analysis was performed using the CIRIfull and KNIFE algorithms, and circRNAs identified with both algorithms were subjected to differential expression (DE) analysis and validation by qPCR. Circ-RCAN2 and circ-C12orf29 expressions were significantly downregulated in infarcted tissue compared to healthy pig heart. Sanger sequencing was performed to identify the backsplice junctions of circular transcripts. Finally, we compared the expressions of circ-C12orf29 and circ-RCAN2 between porcine cardiac progenitor cells (pCPCs) that were incubated in a hypoxia chamber for different time periods versus normoxic pCPCs. Circ-C12orf29 did not show significant DE in vitro, whereas circ-RCAN2 exhibited significant ischemia-time-dependent upregulation in hypoxic pCPCs. Overall, our results revealed novel cardiac circRNAs with DE patterns in pCPCs, and in infarcted and healthy myocardium. Circ-RCAN2 exhibited differential regulation by myocardial infarction in vivo and by hypoxia in vitro. These results will improve our understanding of circRNA regulation during acute MI.

scholarly journals In-vitro culture system for mesenchymal progenitor cells derived from waste human ovarian follicular fluid

2014 ◽  
Vol 29 (4) ◽  
pp. 457-469 ◽  
Author(s):  
Federica Riva ◽  
Claudia Omes ◽  
Roberto Bassani ◽  
Rossella E Nappi ◽  
Giuliano Mazzini ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Follicular Fluid ◽  
Culture System ◽  
Mesenchymal Progenitor Cells ◽  
In Vitro Culture System ◽  
Ovarian Follicular Fluid

Abstract 18845: Induced Cardiac Progenitor Cells Differentiate Into Cardiac Lineage Cells in vivo and Improve Survival in Mice post Myocardial Infarction

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Pratik A Lalit ◽  
Max R Salick ◽  
Daryl O Nelson ◽  
Jayne M Squirrell ◽  
Christina M Shafer ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Disease Modeling ◽  
Ex Vivo ◽  
Cardiac Progenitor Cells ◽  
Heart Tube ◽  
Whole Embryo Culture ◽  
Cardiac Actin ◽  
Cardiac Lineage

Several studies have reported reprogramming of fibroblasts (Fibs) to induced cardiomyocytes, and we have recently reprogrammed mouse Fibs to induced cardiac progenitor cells (iCPCs), which may be more favorable for cardiac repair because of their expandability and multipotency. Adult cardiac (AC), lung and tail-tip Fibs from an Nkx2.5-EYFP reporter mouse were reprogrammed using a combination of five defined factors into iCPCs. Transcriptome and immunocytochemistry analysis revealed that iCPCs were cardiac mesoderm-restricted progenitors that expressed CPC markers including Nkx2.5, Gata4, Irx4, Tbx5, Cxcr4, Flk1 etc. iCPCs could be extensively expanded (over 30 passages) while maintaining multipotency to differentiate in vitro into cardiac lineage cells including cardiomyocytes (CMs), smooth muscle cells and endothelial cells. iCPC derived CMs upon co-culture with mESC-derived CMs formed intercellular gap junctions, exhibited calcium transients, and contractions. The purpose of this study was to determine the in vivo potency of iCPCs. Given that the Nkx2.5-EYFP reporter identifies embryonic CPCs, we first tested the embryonic potency of iCPCs using an ex vivo whole embryo culture model injecting cells into the cardiac crescent (CC) of E8.5 mouse embryos and culturing for 24 to 48 hours. GFP labeled AC Fibs were first tested and live imaging revealed that after 24 hours these cells were rejected from the embryo proper and localized to the ecto-placental cone. In contrast, iCPCs reprogrammed from AC Fibs when injected into the CC localized to the developing heart tube and differentiated into MLC2v, αMHC and cardiac actin expressing CMs. Further we injected iCPCs into infarcted adult mouse hearts and determined their regenerative potential after 1-4 wks. The iCPCs significantly improved survival (p<0.01 Mantel-Cox test) in treated animals (75%) as compared to control (11%). Immunohistochemistry revealed that injected iCPCs localized to the scar area and differentiated into cardiac lineage cells including CMs (cardiac actin). These results indicate that lineage reprogramming of adult somatic cells into iCPCs provides a scalable cell source for cardiac regenerative therapy as well as drug discovery and disease modeling.

Abstract P014: Oxidative Stress Mediated Regulation of Antioxidants in Cardiac Progenitor Cells

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Gokulakrishnan Iyer ◽  
Michael E Davis
Keyword(s):  
Oxidative Stress ◽  
Xanthine Oxidase ◽  
Progenitor Cells ◽  
Cardiac Progenitor Cells ◽  
Dependent Manner ◽  
Akt Inhibitor ◽  
Differentiation Potential ◽  
Lineage Differentiation ◽  
Phosphorylated Akt

Cardiac diseases are the leading causes of death throughout the world and transplantation of endogenous myocardial progenitor population with robust cardiovascular lineage differentiation potential is a promising therapeutic strategy. Therefore, in vitro expansion and transplantation of cardiac progenitor cells (CPCs) is currently in early clinical testing as a potential treatment for severe cardiac dysfunction. However, poor survival and engraftment of cells is one of the major limitations of cell transplantation therapy. Oxidative stress is increased in the ischemic myocardium and indirect inferences suggest the vulnerability of CPCs to oxidative stress. In this study, we show that in vitro, resident c-kit positive CPCs isolated from rat myocardium are significantly (p<0.05) resistant to superoxide-induced apoptosis compared to cardiomyocytes as analyzed by the number of sub-G1 population following xanthine/xanthine oxidase treatment. Interestingly, CPCs have two to four fold higher basal SOD1 and SOD2 activities (p<0.01) compared to cardiomyocytes and endothelial cells. Superoxide treatment increased expression of SOD1 (p<0.01), SOD2 (p<0.01), and glutathione peroxidase (p<0.05) mRNAs within 6 h of treatment compared to control cells. Recent studies suggest the involvement of AKT in controlling cell death, survival and also expression of SOD enzymes. Therefore, we investigated the involvement of AKT in CPCs subjected to oxidative stress. Western blot analysis revealed that the amount of phosphorylated AKT increased significantly within 10 minutes of xanthine/xanthine oxidase treatment. In addition, treatment with LY294002 - a PI3 kinase/AKT inhibitor, increased apoptosis in CPCs treated with superoxide. Our studies demonstrate a novel finding in which resident progenitor cells are protected from oxidative injury by containing higher basal levels of antioxidants as compared to myocytes. Moreover, under oxidant challenge antioxidant levels are regulated, possibly in an AKT-dependent manner. Further elucidation of this pathway may lead to novel therapeutic opportunities.

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.

In Vitro Chemosensitivity of Leukaemic Stem and Progenitor Cells to Gemtuzumab Ozogamicin (Mylotarg) in AML.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 650-650
Author(s):  
Mays Jawad ◽  
Ullas Mony ◽  
Nigel H. Russell ◽  
Monica Pallis
Keyword(s):  
In Vitro Culture ◽  
Progenitor Cells ◽  
Preliminary Data ◽  
Gemtuzumab Ozogamicin ◽  
Flow Cytometric Assay ◽  
Total Percentage ◽  
Flow Cytometric ◽  
Cell Kill ◽  
Stem And Progenitor Cells

Abstract Preliminary data from 1115 patients entered into the MRC AML 15 trial indicated that the addition of Gemtuzumab Ozogamicin (GO) to induction chemotherapy improved disease free survival (Abstract #13, ASH 2006). We hypothesised that this improved survival may be underpinned by the specific therapeutic targeting of leukaemic stem and progenitor cells (LSPC). The LSPC subset of AML cells contains those cells capable of self-renewal in culture and of recapitulating leukaemia in animal models. Successful chemotherapeutic targeting of this subset is essential for complete eradication of leukaemia. We have devised a flow cytometric assay which allows us to measure the in vitro chemosensitivity of the LSPC (CD34+CD38-CD123+) subset in as few as 100 cells and we have used the assay to screen the effectiveness of GO against LSPC. CD123 expression is a determining cell surface marker for leukaemic versus normal stem cells and we were able to demonstrate a significant difference in CD123 MFI values between CD34+CD38- of leukaemic (n= 16) versus normal CD34+ CD38- cells (n= 5; p=0.03), demonstrating the sensitivity of our flow cytometric assay in detecting this leukaemic subset. Blast cells from 14 AML samples were treated with GO (10ng/ml) for 48 hours in an in vitro culture system that maintains LSPC viability. A significant reduction in the number of LSPC (n=14; median 46% cell kill; p= 0.002) as well as AML bulk cells (n=14; median 16% cell kill; p= 0.005) was achieved. This data demonstrates the chemosensitivity of AML cells to GO, particularly to the LSPC subset (p=0.001). Also, the total percentage of LSPC at the start of the assay was found to be positively correlated with GO chemosensitivity (p<0.0001) at 48 hours in in vitro culture (n=14). We have extended culture time for up to 96 hours and preliminary data suggest a further achievable LSPC kill (median 51% cell kill; n= 8). CD33 expression in bulk and CD34+ CD38- populations was explored in the same AML patients. Although CD33 MFI values were highly variable (n= 16; Median = 34.82 and range= 3.7 – 116.54 in bulk fraction and median = 13.69 and range= 0.47 – 436.73 in CD34+ CD38- fraction), we found a significant correlation in CD33 MFI values between bulk and CD34+ CD38- cells (p< 0.0001). Also, the total percentage of CD34+CD38-CD33+ cells was found to be positively correlated with LSPC GO chemosensitivity (n= 14; p= 0.04) after 48 hours of in vitro culture. The GO chemosensitivity of mononuclear cells from mobilised healthy donors was investigated and these were found to be insensitive to this agent both at the bulk cell level and in the CD34+ CD38- subset (mean % cell kill of 10% and 5%, respectively; n=3) after 48 hour in vitro culture. This data establishes the specific targeting of GO to CD123+ CD34+ CD38- and CD33+CD34+ CD38- LSPC, while sparing normal stem and progenitor cells. In conclusion, with many novel agents and drug combinations available for research, we have developed an assay for screening drug effectiveness against LSPC and have demonstrated that GO targets this subset effectively. Combination drugs with GO now need to be further investigated for the complete eradication of LSPC.

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