fellows only session (fos) 2015...may 15, 2010 · fellows only session (fos) 2015 2 welcome...

PCBC Fellows Only Session (FOS) Monday, October 12, 2015

Houston Methodist Research Institute

Research Institute Building (RIB) Board Room/ R2-311, 2nd Floor

Fellows Only Session (FOS) 2015

www.progenitorcells.org

Table of Contents Welcome .......................................................................................................................................... 2 Agenda ............................................................................................................................................ 3 ABSTRACT # 1 - Bhairab Singh (Garry Hub Site 16) Etv2-miR-130a Network Regulates Mesodermal Specification .................................................... 6 ABSTRACT # 2 - Sergii Kyrychenko (Schneider Hub Site 12)

Stem Cell Aliens .......................................................................................................................... 7 ABSTRACT # 3 - Eyayu Belay (Torok-Storb Hub Site 05)

CD34+ Cell Derived In Vitro Erythroid Islands Produce Large Number of Orthochromatic Normoblasts………………………...………………………………………….…..….. 8

ABSTRACT # 4 - Joseph Mandelbaum (Daley Hub Site 08) Chemical Screening Using Zebrafish Embryo Cultures Identifies Adenosine as a Modulator of Hematopoietic Stem and Progenitor Cell Induction……………………………... 9

ABSTRACT # 5 - Amie Adams (Torok-Storb Hub Site 05) 3D Marrow Microenvironment Demonstrates Importance of Endothelial Cells for Megakaryocyte Migration Needed for Platelet Release……………………………………10

ABSTRACT # 6 - Joe Ouadah (Krasnow Hub Site 06) The Cellular and Molecular Origins of Small Cell Lung Cancer………………………………….... 11

ABSTRACT # 7 - Gianfranco Matrone (Cooke Hub Site 10) Discovery and Characterization of a Novel Transcription Factor for Endothelial Cell Development and Lineage Specification……………………………..………….12

ABSTRACT # 8 - Libang Yang (Garry Hub Site 16) Cardiac Smooth Muscle Cell Defined Differentiation of Human Induced Pluripotent Stem Cells…………………………………………………….…………………………..…13

ABSTRACT # 9 - Pratik Lalit (Thomson Hub Site 2) Induced Cardiac Progenitor Cells Differentiate into Cardiac Lineage Cells In Vivo and Improve Survival in Mice Post Myocardial Infarction…………………….….......14

ABSTRACT # 10 - Gautham Yepuri (Cooke Hub Site 10) Proton Pump Inhibitors Impair Vascular Function by Accelerating Endothelial Senescence ............................................................................................................ 15

ABSTRACT # 11 - Ioannis Karakikes (Wu Hub Site 04) Genetic Correction of a TNNT2 Mutation in Human iPSCs Links Dilated Cardiomyopathy to Troponin T Gene Isoform Switching……………………….……16

ABSTRACT # 12 - Young Wook Chun (Hatzopoulos Hub Site 11) Combinatorial Polymer Matrices Enhancing Cardiac Maturation of Human Induced Pluripotent Stem Cells……………………………………...………..………...... 17

ABSTRACT # 13 - Shuangtao Ma (Cooke Hub Site 10) E-selectin-targeting Delivery of MicroRNAs by Microparticles Ameliorates Endothelial Inflammation and Atherosclerosis……. ………………………………..…18

Campus Map and Walking Directions ............................................................................................ 19 Taxi Service ................................................................................................................................... 21 Notes ............................................................................................................................................. 22 Institutional Supporters .................................................................................................................. 29

http://www.progenitorcells.org/


www.progenitorcells.org 2

Welcome

October 12, 2015

Dear Colleagues and Friends,

Welcome to the Houston Methodist Research Institute and the 7th Annual Meeting of the NHLBI Progenitor Cell Biology Consortium (PCBC). We will celebrate our achievements during the past 6 years, and extend our collegial and collaborative interactions as we review the past year’s progress and look forward to the future of the PCBC.

We have an exciting agenda, starting with the Fellows Only Session to highlight the work of our postdoctoral fellows and graduate students. The following two days will include Research Hub reports, breakout sessions, and reports from PCBC task forces and committees. The poster session will give attendees the opportunity to learn about the broad spectrum of the Consortium’s work in stem cell biology and regenerative medicine. Each Research Hub will showcase their novel methodologies and expertise at the Technology Fair. And to help us celebrate our years together, join us for some down-home entertainment Tuesday night!

Our meeting is in the Texas Medical Center (TMC), the largest medical complex in the world. TMC is within walking distance of Rice University, Hermann Park, and the Houston Zoo. If you want to extend your expedition, just north of Hermann Park is the Museum District, 19 museums which include The Museum of Fine Arts, one of the largest museums in the U.S. (a permanent collection that spans more than 6,000 years of history with 64,000 works from six continents). The Houston Museum of Natural Science is one of the most popular in the U.S., with a wonderful dinosaur exhibit (my favorite), but don’t miss the Cockrell Butterfly Center, a transcendent experience with iridescent butterflies flitting through a tropical jungle.

This meeting would not have been possible without the support of our sponsors, the leadership of Dr. Denis Buxton, the assistance of Dr. Michael Terrin and his team, and the diligence of Ayesha Williams at Houston Methodist. Thank you to all attendees for your participation, and your innovation over the past years which has made this Consortium so successful!

With warmest regards,

John P. Cooke, M.D., Ph.D.Joseph C. "Rusty" Walter and Carole Walter Looke Presidential Distinguished Chair in Cardiovascular Disease ResearchDirector, Center for Cardiovascular RegenerationChair, Department of Cardiovascular Sciences Houston Methodist Research Institute

John P. Cooke, M.D., Ph.D.Joseph C. "Rusty" Walter and Carole Walter Looke Presidential Distinguished Chair in Cardiovascular Disease ResearchDirector, Center for Cardiovascular RegenerationChair, Department of Cardiovascular Sciences Houston Methodist Research Institute6670 Bertner Ave, Houston TX 77030Mail Stop: R10-SouthHouston, Texas 77030




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Agenda 7th Annual NHLBI PCBC Fellows Only Session (FOS)

Monday, October 12, 2015 Houston Methodist Research Institute, Houston, TX

08:00 AM Registration and Breakfast RIB Second Floor Mezzanine/RI Boardroom

08:30 AM Welcome and Opening Remarks Shu Meng Fellows Research Reports

08:40 AM Bhairab Singh, Hub Site 16 “Etv2-miR-130a Network Regulates Mesodermal Specification”

09:00 AM Sergii Kyrychenko, Hub Site 12 Stem Cell Aliens

09:20 AM Eyayu Belay, Hub Site 5 “CD34+ Cell Derived In Vitro Erythroid Islands Produce Large Number of Orthochromatic Normoblasts”

09:40 AM Joseph Mandelbaum, Hub Site 8 “Chemical Screening Using Zebrafish Embryo Cultures Identifies Adenosine as a Modulator of Hematopoietic Stem and Progenitor Cell Induction”

10:00 AM Break 10:20 AM Amie Adams, Hub Site 5

“3D Marrow Microenvironment Demonstrates Importance of Endothelial Cells for Megakaryocyte Migration Needed for Platelet Release”

10:40 AM Joe Ouadah, Hub Site 6 “The Cellular and Molecular Origins of Small Cell Lung Cancer”

11:00 AM Special Talk: Clinical Development of mRNA-Based Therapeutic Approaches Dr. Ivone Bruno 11:45 AM Lunch RIB Second Floor Mezzanine/RI Boardroom

12:45 PM Career Development and NIH Grant Mechanisms Drs. Beverly Torok-Storb and Mark Roltsch

1:45 PM Bioinformatics Session




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2:45 PM Gianfranco Matrone, Hub Site 10 “Discovery and Characterization of a Novel Transcription Factor for Endothelial Cell Development and Lineage Specification”

3:05 PM Libang Yang, Hub Site 16 “Cardiac Smooth Muscle Cell Defined Differentiation of Human Induced

Pluripotent Stem Cells”

3:25 PM Pratik Lalit, Hub Site 2 “Induced Cardiac Progenitor Cells Differentiate into Cardiac Lineage Cells In Vivo and Improve Survival in Mice Post Myocardial Infarction”

3:45 PM Break

4:05 PM Gautham Yepuri, Hub Site 10 “Proton Pump Inhibitors Impair Vascular Function by Accelerating Endothelial Senescence”

4:25 PM Ioannis Karakikes, Hub Site 4 “Genetic Correction of a TNNT2 Mutation in Human iPSCs Links Dilated Cardiomyopathy to Troponin T Gene Isoform Switching”

4:45 PM Young Wook Chun, Hub Site 11 “Combinatorial Polymer Matrices Enhancing Cardiac Maturation of Human Induced Pluripotent Stem Cells”

5:05 PM Shuangtao Ma, Hub Site 10 “E-selectin-targeting Delivery of MicroRNAs by Microparticles Ameliorates Endothelial Inflammation and Atherosclerosis”

6:00-9:00 PM Social Event/Dinner TMC Boardroom/Trevisio Restaurant/Cocktail Reception




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Abstracts




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ABSTRACT # 1

Etv2-miR-130a Network Regulates Mesodermal Specification

Bhairab N. Singh, Yasuhiko Kawakami, Ryutaro Akiyama, Tara L. Rasmussen, Mary G. Garry, Wuming Gong, Satyabrata Das, Xiaozhong Shi, Naoko Koyano-Nakagawa and

Daniel J. Garry

University of Minnesota, Minneapolis, MN MicroRNAs are known to regulate critical developmental stages during embryogenesis. Here, we defined a novel Etv2-miR-130a cascade that regulates mesodermal specification and determination. Ablation of Dicer in the Etv2-expressing precursors resulted in altered mesodermal lineages and embryonic lethality by E12.5. We identified miR-130a as a direct target of Etv2 and demonstrated its role in the segregation of bipotent hemato-endothelial progenitors towards the endothelial lineage. Gain-of-function experiments demonstrated that miR-130a promoted the endothelial program at the expense of the cardiac program without impacting the hematopoietic lineages. In contrast, CRISPR/Cas9-mediated knockout of miR-130a demonstrated a reduction of the endothelial program without any impact on the hematopoietic lineages. Mechanistically, miR-130a directly suppresses Pdgfra expression and promotes the endothelial program by blocking Pdgfra signaling. Inhibition or activation of Pdgfra signaling phenocopied the miR-130a over-expression and knockout, phenotype, respectively. This is the first report of a miRNA that specifically promotes the divergence of the common hemato-endothelial progenitor to the endothelial lineage.




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ABSTRACT # 2

Stem Cell Aliens

Sergii Kyrychenko, Sean Goetsch and Jay W. Schneider

UT Southwestern Medical Center, Dallas, TX Small molecules are attractive tools for fine-tuning human pluripotent stem cell (PSC) fate decisions and differentiation programs, and these compounds may have direct translational importance in the near term. Yet, small molecules are often naively expected to have lofty biological activities like encoding a desired cell fate. We have been studying a small molecule known as ISX9, a 3, 5-disubstituted isoxazole originally identified in a pro-cardiogenic small molecule screen (NKX2-5 gene activation) in P19 embryonal carcinoma cells but then later shown to be powerfully pro-neurogenic in mouse neural stem cells and ESCs, and even in vivo in the mouse hippocampus. Neither ISX9’s direct target nor mechanism of action is firmly established. We studied ISX9’s effects on standard GiWi chemical programming of cardiogenesis in human PSCs expecting this molecule to help drive cardiac differentiation, but to our surprise we found that ISX9 completely blocked cardiac differentiation, diverting these cells instead towards an as yet undefined synthetic lineage and phenotype that is probably completely alien to mammalian life as we know it. Among other genes, ISX9 strongly and broadly activated human homeotic (HOX) gene clusters, including HOX A, HOX B, HOX C and HOX D, which encode nodal transcription factors responsible for many aspects of early development and patterning like morphogenesis, cell adhesion, cell division rates, cell death and movement. In some cases ISX9 induced HOX gene expression more than 100-fold at the gene transcript level. These results lead us to speculate more generally that while the current focus is on optimizing small molecule based protocols to generate crucial cells like cardiomyocytes required for regenerative medicine, there will also be high value in alien cells generated by design with small molecules like ISX9 that have unexpected and unnatural or aberrant functions. These studies will also lead to identification of ISX’s target and mechanism of action.




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ABSTRACT # 3

CD34+ Cell Derived In Vitro Erythroid Islands Produce Large Number of Orthochromatic Normoblasts

Eyayu Belay, David W. Emery and Beverly Torok-Storb

Fred Hutchinson Cancer Research Center, Seattle, WA

Erythroid island associated in vivo erythropoiesis is highly efficient but mimicking the system in vitro remains elusive. Recently, we developed a small molecule responsive hyperactive Mpl- based Cell Growth Switch (CGS) that drives macrophage associated erythropoiesis in the absence of exogenous cytokines. Here, we explore the physical, cellular and molecular interaction between the macrophages and erythroid cells in CGS expanded cord blood CD34+ cells. Scanning electron and light microscopy revealed a patterned direct association of erythroblasts to a central macrophage mimicking the bone marrow islands. Gene expression and proteomics studies detected erythroid island associated molecules such as erythroblast macrophage protein (EMP), Intercellular Adhesion Molecule 4 (ICAM-4) and Ephrin type-B receptor 4 (EphB4). The islands also express Ferroportine indicative of iron channeling. Interestingly, addition of conditioned medium from cultures of HS27a marrow fibroblasts lead to a 700 and 90 fold increase in total and erythroid cell number respectively compared to non- treated cells (p=0.0004). In addition, 80 % of the CGS generated erythroblasts were orthochromatic normoblasts, a pre reticulocyte stage of erythroid differentiation, ready to be enucleated within 14 days of culture. The consistent production of these in vitro generated erythroid islands provides a model for identifying the molecular basis of the enucleation process.




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ABSTRACT # 4

Chemical Screening Using Zebrafish Embryo Cultures Identifies Adenosine as a Modulator of Hematopoietic Stem and Progenitor Cell Induction

Joseph Mandelbaum, Ilya Shestopalov, Lili Jing, and Leonard I. Zon

Boston Children’s Hospital, Boston, MA

Hematopoietic stem cells (HSCs) have been used clinically to reconstitute the bone marrow, but half of all patients lack a matched donor marrow. iPSC-derived HSCs hold great promise for treating these patients, but our understanding of the pathways involved in HSC formation is incomplete. We previously reported a chemical genetic screen that identified inducers of myogenesis using an embryonic blastomere culture system in zebrafish (Xu et al., Cell, 2013). Here, we have adapted this culture screening system to find inducers of c-myb, a marker of hematopoietic stem and progenitor cells (HSPCs), to identify new pathways that expand HSPCs. In this study, c-myb:GFP zebrafish are grown to 50% epiboly and dissociated into single cells, which are then plated in 384 well plates. The cultured cells are treated with 3,840 independent compounds in duplicate, and after two days in culture, GFP fluorescence is read out. We identified 14 chemicals (z score >10; hit rate 0.36%) that induce c-myb expressing cell expansion and performed dose response curves. Among these hits was the adenosine analogue 3-deazaadenosine, suggesting adenosine regulates HSPC formation. We treated whole embryos with different adenosine analogues, including the adenosine receptor agonist 5’-N-ethylcarboxamidoadenosine (NECA) and antagonist CGS15943, to examine effects on HSPC development. Confocal imaging of the aorta-gonad-mesonephros (AGM) region, the site where HSPCs are specified, showed a significantly increased number of HSPCs in cmyb:GFP embryos (52.8±4.8) vs DMSO control (43.2±2.4) due to NECA treatment, and significantly decreased (36.0±2.4) due to CGS15943 treatment. Since HSPCs originate from hemogenic endothelium by undergoing endothelial-to-hematopoietic transition (EHT), we wanted to observe whether adenosine has an effect on endothelial cells by time-lapse imaging. Morpholino A2b receptor injections to knockdown this adenosine receptor caused more endothelial cells, marked by flk1:GFP, to abort EHT. Taken together, we conclude that adenosine signaling is required for EHT and proper HSPC development. Factors revealed in our zebrafish culture system establish new pathways that can be useful targets to promote iPSC-derived HSPC expansion; our findings identify an important role of adenosine signaling in HSPC emergence.




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ABSTRACT # 5

3D Marrow Microenvironment Demonstrates Importance of Endothelial Cells for Megakaryocyte Migration Needed for Platelet Release

Amie Adams, Surya Kotha, Sijie Sun, Beverly Torok-Storb and Ying Zheng

University of Washington, Seattle, WA

To test the hypothesis that megakaryocyte (MK) migration required for platelet production and release is influenced by endothelial cells (ECs), we created an ex vivo 3D bone marrow microenvironment (ME). First, MKs differentiated from human CD34+ and ECs were co-cultured in a 3D collagen matrix under static conditions for ten days; MKs were embedded in type I collagen with a top EC monolayer. The MK density was quantified with respect to the distance from the ECs using IHC and confocal microscopy at designated time points. We observed a significant accumulation of MKs within 50 microns of an endothelial monolayer. In contrast, in the absence of EC monolayer at the top there was no accumulation of MKs. Second, MKs and ECs were co-cultured in a flow-mediated thrombopoietic vascular ME in vitro, where a 3D microvascular network was fabricated using soft lithographic technique and injection molding of MKs embedded collagen. MKs showed active migration to the vessel wall as detected by time course quantification and live imaging. Mature MKs penetrated the endothelial layer and released pro-platelets and platelets into the vessel lumen. Immature MKs also penetrated the endothelium and entered the circulation. Introduction of anti-CXCR4 antibody to the system blocked MK migration. Our data suggest that ECs attract MKs via CXCR4-CXCL12 signaling regardless of their maturation.




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ABSTRACT # 6

The Cellular and Molecular Origins of Small Cell Lung Cancer

Joe Ouadah, Dan Riordan, and Mark Krasnow

Stanford University, Stanford, CA Small cell lung cancer originates from pulmonary neuroendocrine cells (PNECs), evolutionarily conserved neurosensory cells that are a rare constituent of the vertebrate airway epithelium. Unlike many other known cancer cell types of origin, adult PNECs are quiescent during normal homeostasis and therefore presumed to be terminally differentiated. However, at least under conditions of severe epithelial injury, PNECs contribute to lung regeneration and thus are considered 'facultative' progenitors or stem cells. How is PNEC proliferation and reprogramming controlled and coordinated? How are the control mechanisms altered by the combination of mutations and carcinogen exposure that is characteristic of small cell lung cancer? We are using mouse genetics, injury models, and single-cell RNA-sequencing to elucidate the cellular and molecular controls of PNEC proliferation and reprogramming, to define the normal control pathways, how they go awry in small cell lung cancer, and to provide a framework for someday rationally treating and preventing tumor initiation. We will describe progress toward the characterization of PNEC regeneration at single cell resolution and insights into the genetic bases of PNEC proliferation and reprogramming, and the implications for small cell lung cancer.




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ABSTRACT # 7

Discovery and Characterization of a Novel Transcription Factor for Endothelial Cell Development and Lineage Specification

Gianfranco Matrone, Xiao Yu Tian, Helen Blau, Wing T. Wong and John P. Cooke

Houston Methodist Research Institute, Houston, TX

Background: We have developed bi-species heterokaryons [generated by fusion of mouse embryonic stem cells (mESC) and human endothelial cells (hEC) as a model system for discovery of novel factors required for endothelial lineage. Our RNAseq data suggests that the determinants of endothelial phenotype in the hEC act on the mESC to recapitulate endothelial ontogeny. Furthermore, novel transcription factors (TF) in endothelial specification were implicated, such as POU domain-containing TF (POU3F2), also called BRN2 or N-Oct3. This study assessed the role of POU3F2 in endothelial cell differentiation and in zebrafish vascular development. Methods: EC differentiation was induced by culture of mESC with growth factors (VEGF, bFGF and BMP4). POU3F2 loss-of-function was induced by lentiviral shRNA in mESCs. FACS was used to analyze cell lineage. Tg (Fli1a: GFP) zebrafish were used to analyze vascular phenotype. For POU3F2 knockdown in zebrafish, caged morpholino was injected in embryos at 1-2 cell stage and activated at 6 or 24 hour post fertilization by embryo exposure to UV light. Zebrafish GFP+ cells were enzymatically purified from embryos and quantified by FACS. In-situ hybridization with RNA sense (control) and antisense probes was used to localize POU3f2 mRNA in the zebrafish embryo. Real time PCR and western blotting were used for a semi-quantitative analysis of gene and protein expression respectively. Results: POU3F2 knockdown in mESCs reduced the Flk1+CD144+ cell population during differentiation of mESCs. POU3F2 knockdown also reduced endothelial cell markers in this mESC derived Flk1+CD144+ cells, including Kdr, Cdh5, Nos3, Tie2 and Lmo2; and reduced EC tube formation in matrigel. In zebrafish embryos, injection of MOs targeting POU3F2 reduced POU3F2 mRNA localization, detected by in-situ hybridization, protein at 24 and 48 hpf, and GFP+ cells. This was associated with an embryo phenotype characterized by severe vascular aberrations. Conclusions: Our heterokaryon studies implicated the transcription factor POU3F2 in endothelial cell development. We validated the role of POU3F2 in the reprogramming of pluripotent stem cells to EC lineage. We provide data that POU3F2 is required for normal vascular development in the zebrafish.




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ABSTRACT # 8

Cardiac Smooth Muscle Cell Defined Differentiation of Human Induced Pluripotent Stem Cells

Libang Yang and Jianyi Zhang

University of Minnesota, Minneapolis, MN

Rationale and Objective: Smooth muscle cells (SMCs) are found in blood vessels, lymphatic vessels, the urinary bladder, uterus, reproductive tracts, gastrointestinal tract, respiratory tract, etc. HiPSs can be differentiated into cells of several lineages, including SMCs, which can be useful for testing new drugs, modelling disease in above tissue and may prove to be an effective treatment for patients with diseases in above tissues. However, current SMC differentiation protocols can be inefficient, and the phenotypes of the differentiated SMCs are not easy to be defined and purified, which inhibits their utility for basic science and therapy research. This report introduces a novel and remarkably more efficient hiPS-SMC differentiation protocol. Methods and Results two of 2~3 weeks protocols were developed to differentiate hiPS into two subtype of SMCs, up to 50% of the differentiated hiPSs assumed an SMC phenotype, and after purification to >90% (based on SMA expression), the HiPS-SMCs are stable to display SMC characteristics for more than 4 months and the subtype of SMC could be control in vitro. Gene and protein expression levels of SMA, SM22, collagen I, and connexion 43 was significantly upregulated in the differentiated HiPS-SMCs; SMA, SM22 were highly expressed but not collagen I and connexin 43 in contractile SMC. SMC biological function was demonstrated via the measurement of contraction, migration and cell proliferation of subtypes of SMCs. The con-SMC had higher contraction ability. In contraction test with Carbachol treatment, Contractile-SMC area reduced to 12% of original size while synthetic-SMC shrunk to 44.3% of their original size. In scratch test for measurement of migration, 12 hours after scratch, more synthetic -SMC occupy the scratched empty space while contractile -SMC only few cells moved to the scratched area (Synthetic -SMC with 36±5.2 vs contractile -SMC with 6±4.2 per field). Synthetic-SMC also shown stronger proliferation ability when synthetic-SMC and contractile-SMC treated with PDGFbβ and TGFβ. Conclusion: These data demonstrate two robust xeno free, efficient and reagent defined SMC differentiation protocol in vitro. Differentiated SMCs are stable for more than 4 months.




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ABSTRACT # 9

Induced Cardiac Progenitor Cells Differentiate into Cardiac Lineage Cells in Vivo and Improve Survival in Mice Post Myocardial Infarction

Pratik A. Lalit, Max R. Salick, Christina M. Shafer, Michael Kyba, Daniel J. Garry, Ron Stewart,

James A. Thomson, Karen M. Downs and Timothy J. Kamp

University of Wisconsin-Madison, Madison, WI We recently reprogrammed mouse fibroblasts (Fibs) into induced cardiac progenitor cells (iCPCs), which may be 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 (SMs) and endothelial cells (ECs). iCPC-CMs upon co-culture with mESC-CMs exhibited calcium transients and contractions. The purpose of this study was to determine the in vivo potency of iCPCs. 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 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 engrafted within the scar area and differentiated into cardiac lineage cells including CMs (cardiac actin) SMs (SMA) and ECs (CD31). 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.




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ABSTRACT # 10

Proton Pump Inhibitors Impair Vascular Function by Accelerating Endothelial Senescence

Gautham Yepuri, Roman Sukhovershin, Timo Z. Nazari-shafti, Yohannes T. Ghebremariam

and John P. Cooke

Houston Methodist Research Institute, Houston, TX Objective: Aging, vascular senescence and endothelial dysfunction are major risk factors for cardiovascular disease (CVD). Proton pump inhibitors (PPIs) like Esomeprazole (Nexium) are extensively used drugs for the treatment of gastroesophageal reflux disease (GERD). Recently, the use of PPI has been associated with increased cardiovascular (CV) risk. In this study, we hypothesized that PPIs pose CV risk by accelerating vascular aging and endothelial dysfunction. Therefore, we aimed to investigate the mechanisms that may lead to the development of CVD. Methods and Results: Long-term incubation of human microvascular endothelial cells (ECs) with clinically relevant concentration of PPIs (esomeprazole) accelerated premature endothelial cell senescence by shortening telomere length. A significant increase in superoxide levels and decrease in nitric oxide production, decrease in DDAH, eNOS and iNOS gene expression were also observed upon PPI treatment. These factors are well known to increase oxidative stress and exacerbate endothelial dysfunction. Functionally, PPIs impaired the angiogenic and proliferative capacity of ECs as confirmed by Matrigel tube formation and cell proliferation assays. Investigation of the molecular pathways involved in PPI-accelerated endothelial senescence revealed that plasminogen activator inhibitor-1(PAI-1); a gene commonly associated with EC senescence and telomere length, was significantly up-regulated by PPI treatment. Furthermore, PPIs downregulated all genes involved in Shelterin complex (a set of genes involved in regulation and maintenance of telomere function and also responsible for the regulation and signaling of DNA damage response pathways) providing a mechanistic evidence for the role of PPIs in accelerating endothelial senescence. Conclusion: Our data provides the first molecular and mechanistic evidence that PPIs pose a major risk for CVDs by accelerating endothelial senescence and endothelial dysfunction through, at least in part, activation of PAI-1 protein and telomere shortening. Given the widespread and long-term use of PPIs in the absence of medical supervision, our data raises a major safety concern.




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ABSTRACT # 11

Genetic Correction of a TNNT2 Mutation in Human iPSCs Links Dilated Cardiomyopathy to Troponin T Gene Isoform Switching

Ioannis Karakikes, Vittavat Termglinchan, Timon Seeger, Mohamed Ameen, Haodi Wu, Elena

Matsa and Joseph C. Wu

Stanford University, Stanford, CA Dilated cardiomyopathy (DCM) is a leading cause of heart failure. Inherited cardiomyopathies, including DCM, have been recently modeled with induced pluripotent stem cells (iPSCs), providing a human-based model to study familial cardiomyopathies in vitro. We hypothesized that combining iPSC-based disease modeling and gene-editing technologies will provide novel insights into the molecular mechanism underlying the pathogenesis of DCM. We derived iPSCs from DCM patients harboring a heterozygous mutation in the cardiac troponin T gene (TNNT2; p.R173W), and generated matched isogenic iPSC clones by correcting the mutation using transcription activator-like effector nucleases (TALENs). We showed that the genome-editing-based correction of the TNNT2 R173W mutation resulted in phenotypic rescue in differentiated cardiomyocytes. Expression profiling of pairs of isogenic wild-type and mutant cardiomyocytes revealed several genes that are consistently dysregulated by the mutant TNNT2 gene, including the fast-twitch skeletal Troponin T (TNNT3) and it’s transcriptional repressor prospero homeobox 1 (PROX1). We demonstrated that Troponin T isoform switching contributes to cardiomyocyte dysfunction and showed that DCM-associated phenotypes were ameliorated by PROX1 gene therapy in vitro, suggesting therapeutic potential. In summary, we demonstrate that gene correction resulted in phenotypic rescue in differentiated cardiomyocytes and uncovered expression changes associated with TNNT2 R173W mutation. Our results provide novel mechanistic insights into the pathogenesis of DCM induced by mutant TNNT2 that could enable the development of new personalized therapeutics.




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ABSTRACT # 12

Combinatorial Polymer Matrices Enhancing Cardiac Maturation of Human Induced Pluripotent Stem Cells

Young Wook Chun, Hak-Joon Sung and Charles C. Hong

Vanderbilt University, Nashville, TN Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo, we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro. In this study, we employed a library of combinatorial polymers comprising of three functional subunits e poly-ε-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-96%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro. Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions.




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ABSTRACT # 13

E-selectin-targeting Delivery of MicroRNAs by Microparticles Ameliorates Endothelial Inflammation and Atherosclerosis

Shuangtao Ma, Xiao Yu Tian, Yunrong Zhang, Chaofeng Mu, Haifa Shen, Jean Bismuth,

Yu Huang, Wing Tak Wong

Houston Methodist Research Institute, Houston, TX E-selectin serves as a surface marker of inflamed endothelial cells (ECs). We attempted to deliver the therapeutic agents to inflamed endothelium covering atherosclerotic plaques with an E-selectin-targeting multistage vector (ESTA-MSV), a microparticle that we developed previously. To test the hypothesis whether E-selectin-targeting delivery of microRNA (miR)-146a and miR-181b, which hold potentials to inhibit adhesion molecules, ameliorates endothelial inflammation and atherosclerosis. Cy5-conjugated miR-146a and miR-181b were packaged in polyethylene glycol-polyethyleneimine (PEG/PEI) nanoparticles and then loaded into ESTA-MSV microparticles. The expression of both miRs was downregulated in tumor necrosis factor (TNF)-α-treated ECs. We transfected TNF-α-treated mouse aortas and cultured human ECs and found that the transfection efficiency of ESTA-MSV to deliver miRNA was significantly higher than PEG/PEI. The expression of adhesion molecules, including chemokines CCL2, CCL5, CCL8, and CXCL9, and the adhesion of THP-1 monocytes to ECs were suppressed by miR-146a/-181b packaged in ESTA-MSV. The apolipoprotein E-deficient mice were fed with Western diet and injected intravenously with the particles prepared as above biweekly for 12 weeks. Atherosclerotic plaque size decreased with treatment of miRs packaged in ESTA-MSV but not in PEG/PEI. Moreover, vascular inflammation markers such as macrophages in aortic root lesion and expression of adhesion molecules in aortic tissues were decreased while the vascular smooth muscle cells and collagen were increased in plaques from ESTA-MSV/miRs-treated mice compared with vehicle-treated group. Our data demonstrate that the ESTA-MSV microparticle-mediated specific and efficient delivery of miR-146a/-181b to the inflamed endothelium ameliorates the endothelial inflammation and atherosclerosis.




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Campus Map




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Walking Directions:

• The Fellows Only Session will be held in the RI Boardroom, 2nd floor, adjacent to the John F. Bookout Auditorium, in the Methodist Hospital Research Institute.




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Taxi Service

Campus Address: Houston Methodist Research Institute RIB Second Floor Mezzanine/ John F. Bookout Auditorium 6670 Bertner Avenue Houston, TX 77030

Hotel Address: Houston Marriott Medical Center 6580 Fannin Street Houston, TX, 77030

Houston Taxi Service 2717 Stoney Brook Dr #201 Houston, TX 77057 (832) 661-5590

Houston Town Car And Limo 710 Smith St Houston, TX 77002 (281) 630-1137

Houston VIP Taxi 1530 Candytuft St Houston, TX 77038 (281) 616-5838

Houston 24 Hour Airport Limo 711 Louisiana St Houston, TX 77002 (832) 421-8002

HTS Limited 5825 Kelley St Houston, TX 77026 (713) 659-5105

Yellow Cab Houston 1406 Hays St Houston, TX 77009 (713) 236-1111


http://www.google.com/imgres?q=taxi+image&sa=X&rlz=1I7RNVA_enUS534&biw=1205&bih=717&tbm=isch&tbnid=lHmh7wZcsWeDrM:&imgrefurl=http://www.les-photos-gratuites.net/wp-content/gallery/taxi/&docid=_64RFJfBEeJpmM&imgurl=http://www.les-photos-gratuites.net/wp-content/gallery/taxi/dessin-taxi.jpg&w=1173&h=717&ei=DmQKUr3DMoe14AOmlYGIAg&zoom=1



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Institutional Supporters

• Houston Methodist Research Institute, Center for Cardiovascular Regeneration

• Texas Medical Center (TMC)

• National Heart, Lung, and Blood Institute (NHLBI)

• University of Maryland, Baltimore


fellows only session (fos) 2015...may 15, 2010 · fellows only session (fos) 2015 2 welcome...

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