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Sample records for stem cell-derived cardiac

  1. Induced Pluripotent Stem Cell-Derived Cardiac Progenitors Differentiate to Cardiomyocytes and Form Biosynthetic Tissues

    PubMed Central

    Chakraborty, Syandan; Chellapan, Malathi; Bursac, Nenad; Leong, Kam W.

    2013-01-01

    The mammalian heart has little capacity to regenerate, and following injury the myocardium is replaced by non-contractile scar tissue. Consequently, increased wall stress and workload on the remaining myocardium leads to chamber dilation, dysfunction, and heart failure. Cell-based therapy with an autologous, epigenetically reprogrammed, and cardiac-committed progenitor cell source could potentially reverse this process by replacing the damaged myocardium with functional tissue. However, it is unclear whether cardiac progenitor cell-derived cardiomyocytes are capable of attaining levels of structural and functional maturity comparable to that of terminally-fated cardiomyocytes. Here, we first describe the derivation of mouse induced pluripotent stem (iPS) cells, which once differentiated allow for the enrichment of Nkx2-5(+) cardiac progenitors, and the cardiomyocyte-specific expression of the red fluorescent protein. We show that the cardiac progenitors are multipotent and capable of differentiating into endothelial cells, smooth muscle cells and cardiomyocytes. Moreover, cardiac progenitor selection corresponds to cKit(+) cell enrichment, while cardiomyocyte cell-lineage commitment is concomitant with dual expression of either cKit/Flk1 or cKit/Sca-1. We proceed to show that the cardiac progenitor-derived cardiomyocytes are capable of forming electrically and mechanically coupled large-scale 2D cell cultures with mature electrophysiological properties. Finally, we examine the cell progenitors’ ability to form electromechanically coherent macroscopic tissues, using a physiologically relevant 3D culture model and demonstrate that following long-term culture the cardiomyocytes align, and form robust electromechanical connections throughout the volume of the biosynthetic tissue construct. We conclude that the iPS cell-derived cardiac progenitors are a robust cell source for tissue engineering applications and a 3D culture platform for pharmacological screening and

  2. Isolation and characterization of embryonic stem cell-derived cardiac Purkinje cells.

    PubMed

    Maass, Karen; Shekhar, Akshay; Lu, Jia; Kang, Guoxin; See, Fiona; Kim, Eugene E; Delgado, Camila; Shen, Steven; Cohen, Lisa; Fishman, Glenn I

    2015-04-01

    The cardiac Purkinje fiber network is composed of highly specialized cardiomyocytes responsible for the synchronous excitation and contraction of the ventricles. Computational modeling, experimental animal studies, and intracardiac electrical recordings from patients with heritable and acquired forms of heart disease suggest that Purkinje cells (PCs) may also serve as critical triggers of life-threatening arrhythmias. Nonetheless, owing to the difficulty in isolating and studying this rare population of cells, the precise role of PC in arrhythmogenesis and the underlying molecular mechanisms responsible for their proarrhythmic behavior are not fully characterized. Conceptually, a stem cell-based model system might facilitate studies of PC-dependent arrhythmia mechanisms and serve as a platform to test novel therapeutics. Here, we describe the generation of murine embryonic stem cells (ESC) harboring pan-cardiomyocyte and PC-specific reporter genes. We demonstrate that the dual reporter gene strategy may be used to identify and isolate the rare ESC-derived PC (ESC-PC) from a mixed population of cardiogenic cells. ESC-PC display transcriptional signatures and functional properties, including action potentials, intracellular calcium cycling, and chronotropic behavior comparable to endogenous PC. Our results suggest that stem-cell derived PC are a feasible new platform for studies of developmental biology, disease pathogenesis, and screening for novel antiarrhythmic therapies. PMID:25524238

  3. Finding the rhythm of sudden cardiac death: new opportunities using induced pluripotent stem cell-derived cardiomyocytes.

    PubMed

    Sallam, Karim; Li, Yingxin; Sager, Philip T; Houser, Steven R; Wu, Joseph C

    2015-06-01

    Sudden cardiac death is a common cause of death in patients with structural heart disease, genetic mutations, or acquired disorders affecting cardiac ion channels. A wide range of platforms exist to model and study disorders associated with sudden cardiac death. Human clinical studies are cumbersome and are thwarted by the extent of investigation that can be performed on human subjects. Animal models are limited by their degree of homology to human cardiac electrophysiology, including ion channel expression. Most commonly used cellular models are cellular transfection models, which are able to mimic the expression of a single-ion channel offering incomplete insight into changes of the action potential profile. Induced pluripotent stem cell-derived cardiomyocytes resemble, but are not identical, adult human cardiomyocytes and provide a new platform for studying arrhythmic disorders leading to sudden cardiac death. A variety of platforms exist to phenotype cellular models, including conventional and automated patch clamp, multielectrode array, and computational modeling. Induced pluripotent stem cell-derived cardiomyocytes have been used to study long QT syndrome, catecholaminergic polymorphic ventricular tachycardia, hypertrophic cardiomyopathy, and other hereditary cardiac disorders. Although induced pluripotent stem cell-derived cardiomyocytes are distinct from adult cardiomyocytes, they provide a robust platform to advance the science and clinical care of sudden cardiac death. PMID:26044252

  4. Native Cardiac Extracellular Matrix Hydrogels for Cultivation of Human Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Freytes, Donald O; O’Neill, John D; Duan-Arnold, Yi; Wrona, Emily; Vunjak-Novakovic, Gordana

    2015-01-01

    Summary Biomaterial scaffolds made of native and synthetic materials are designed to serve as a structural and informational template for cell attachment and tissue formation. The use of native extracellular matrix (ECM) is of special interest for the culture of cardiac stem and progenitor cells due to the presence of intrinsic regulatory factors regulating cardiac function. We describe here how to obtain native ECM hydrogels from porcine hearts for the culture of human embryonic, induced pluripotent, and somatic stem cells for cardiac tissue engineering and regenerative medicine applications. PMID:25070328

  5. Natural cardiac extracellular matrix hydrogels for cultivation of human stem cell-derived cardiomyocytes.

    PubMed

    Freytes, Donald O; O'Neill, John D; Duan-Arnold, Yi; Wrona, Emily A; Vunjak-Novakovic, Gordana

    2014-01-01

    Biomaterial scaffolds made of natural and synthetic materials are designed to serve as a structural and informational template for cell attachment and tissue formation. The use of native extracellular matrix (ECM) is of special interest for the culture of cardiac stem and progenitor cells due to the presence of intrinsic regulatory factors regulating cardiac function. We describe here how to obtain native ECM hydrogels from porcine hearts for the culture of human embryonic, induced pluripotent, and somatic stem cells for cardiac tissue engineering and regenerative medicine applications. PMID:25070328

  6. Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues

    PubMed Central

    Ravenscroft, Stephanie M.; Pointon, Amy; Williams, Awel W.; Cross, Michael J.; Sidaway, James E.

    2016-01-01

    The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca2+ transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca2+ handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity. PMID:27125969

  7. Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues.

    PubMed

    Ravenscroft, Stephanie M; Pointon, Amy; Williams, Awel W; Cross, Michael J; Sidaway, James E

    2016-07-01

    The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca(2+ )transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca(2+ )handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity. PMID:27125969

  8. Teratocarcinomas Arising from Allogeneic Induced Pluripotent Stem Cell-Derived Cardiac Tissue Constructs Provoked Host Immune Rejection in Mice

    PubMed Central

    Kawamura, Ai; Miyagawa, Shigeru; Fukushima, Satsuki; Kawamura, Takuji; Kashiyama, Noriyuki; Ito, Emiko; Watabe, Tadashi; Masuda, Shigeo; Toda, Koichi; Hatazawa, Jun; Morii, Eiichi; Sawa, Yoshiki

    2016-01-01

    Transplantation of induced pluripotent stem cell-derived cardiac tissue constructs is a promising regenerative treatment for cardiac failure: however, its tumourigenic potential is concerning. We hypothesised that the tumourigenic potential may be eliminated by the host immune response after allogeneic cell transplantation. Scaffold-free iPSC-derived cardaic tissue sheets of C57BL/6 mouse origin were transplanted into the cardiac surface of syngeneic C57BL/6 mice and allogeneic BALB/c mice with or without tacrolimus injection. Syngeneic mice and tacrolimus-injected immunosuppressed allogeneic mice formed teratocarcinomas with identical phenotypes, characteristic, and time courses, as assessed by imaging tools including 18F-fluorodeoxyglucose-positron emission tomography. In contrast, temporarily immunosuppressed allogeneic mice, following cessation of tacrolimus injection displayed diminished progression of the teratocarcinoma, accompanied by an accumulation of CD4/CD8-positive T cells, and finally achieved complete elimination of the teratocarcinoma. Our results indicated that malignant teratocarcinomas arising from induced pluripotent stem cell-derived cardiac tissue constructs provoked T cell-related host immune rejection to arrest tumour growth in murine allogeneic transplantation models. PMID:26763872

  9. Design and formulation of functional pluripotent stem cell-derived cardiac microtissues

    PubMed Central

    Thavandiran, Nimalan; Dubois, Nicole; Mikryukov, Alexander; Massé, Stéphane; Beca, Bogdan; Simmons, Craig A.; Deshpande, Vikram S.; McGarry, J. Patrick; Chen, Christopher S.; Nanthakumar, Kumaraswamy; Keller, Gordon M.; Radisic, Milica; Zandstra, Peter W.

    2013-01-01

    Access to robust and information-rich human cardiac tissue models would accelerate drug-based strategies for treating heart disease. Despite significant effort, the generation of high-fidelity adult-like human cardiac tissue analogs remains challenging. We used computational modeling of tissue contraction and assembly mechanics in conjunction with microfabricated constraints to guide the design of aligned and functional 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues that we term cardiac microwires (CMWs). Miniaturization of the platform circumvented the need for tissue vascularization and enabled higher-throughput image-based analysis of CMW drug responsiveness. CMW tissue properties could be tuned using electromechanical stimuli and cell composition. Specifically, controlling self-assembly of 3D tissues in aligned collagen, and pacing with point stimulation electrodes, were found to promote cardiac maturation-associated gene expression and in vivo-like electrical signal propagation. Furthermore, screening a range of hPSC-derived cardiac cell ratios identified that 75% NKX2 Homeobox 5 (NKX2-5)+ cardiomyocytes and 25% Cluster of Differentiation 90 OR (CD90)+ nonmyocytes optimized tissue remodeling dynamics and yielded enhanced structural and functional properties. Finally, we demonstrate the utility of the optimized platform in a tachycardic model of arrhythmogenesis, an aspect of cardiac electrophysiology not previously recapitulated in 3D in vitro hPSC-derived cardiac microtissue models. The design criteria identified with our CMW platform should accelerate the development of predictive in vitro assays of human heart tissue function. PMID:24255110

  10. Mesenchymal stem cell-derived exosomes: A novel potential therapeutic avenue for cardiac regeneration.

    PubMed

    Safari, S; Malekvandfard, F; Babashah, S; Alizadehasl, A; Sadeghizadeh, M; Motavaf, M

    2016-01-01

    Coronary artery diseases (CADs) represent a significant cause of death worldwide. During recent decades the rate of cardiovascular mortality has been declined as a result of modern medicine and surgery. However, despite the fact that cardiac cells, including cardiomyocytes (CMCs), vascular smooth muscle cells (VSMC) and vascular endothelial cells (VEC), can be regenerated by cardiac adult stem cell, the regenerative capacity of these cells are limited and inadequate to functionally regenerate heart damaged tissue. Thus, growth reserve of the heart fails to restore the structural integrity of the myocardium after infarction and healing is associated with scar formation. An explanation for this is that cardiac reside stem cells are present throughout the infarction site but die rapidly by apoptosis. Furthermore, microenvironment surrounding the damage site is not promising for the cells survival and renewal. Hence, recent advances in the stem cell therapy have emerged as an attractive approach to replace the lost cells. In this context, mesenchymal stem cells (MSCs) has considered as one of the most promising candidates for regeneration of cardiac cells, lost upon injury. The regenerative capacity of MSCs has primarily been centered on the hypothesis that these cells would engraft, differentiate and replace damaged cardiac cells. However, experimental and clinical observations so far have failed to establish if this differentiated is considerably relevant to MSCs cardiac regenerative properties. Recent reports have suggested that these therapeutic properties, at least in part, are mediated by paracrine factors released from MSCs. This review provides a concise summary of current evidences supporting the paracrine hypothesis of MSCs. In particular, the scope of this review focuses on the role of MSC-derived exosome (MSC-EXs) as a therapeutic modality for the treatment of CADs, particularly ischemic myocardial dysfunctions. PMID:27453275

  11. Three-Dimensional Adult Cardiac Extracellular Matrix Promotes Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    PubMed

    Fong, Ashley H; Romero-López, Mónica; Heylman, Christopher M; Keating, Mark; Tran, David; Sobrino, Agua; Tran, Anh Q; Pham, Hiep H; Fimbres, Cristhian; Gershon, Paul D; Botvinick, Elliot L; George, Steven C; Hughes, Christopher C W

    2016-08-01

    Pluripotent stem cell-derived cardiomyocytes (CMs) have great potential in the development of new therapies for cardiovascular disease. In particular, human induced pluripotent stem cells (iPSCs) may prove especially advantageous due to their pluripotency, their self-renewal potential, and their ability to create patient-specific cell lines. Unfortunately, pluripotent stem cell-derived CMs are immature, with characteristics more closely resembling fetal CMs than adult CMs, and this immaturity has limited their use in drug screening and cell-based therapies. Extracellular matrix (ECM) influences cellular behavior and maturation, as does the geometry of the environment-two-dimensional (2D) versus three-dimensional (3D). We therefore tested the hypothesis that native cardiac ECM and 3D cultures might enhance the maturation of iPSC-derived CMs in vitro. We demonstrate that maturation of iPSC-derived CMs was enhanced when cells were seeded into a 3D cardiac ECM scaffold, compared with 2D culture. 3D cardiac ECM promoted increased expression of calcium-handling genes, Junctin, CaV1.2, NCX1, HCN4, SERCA2a, Triadin, and CASQ2. Consistent with this, we find that iPSC-derived CMs in 3D adult cardiac ECM show increased calcium signaling (amplitude) and kinetics (maximum upstroke and downstroke) compared with cells in 2D. Cells in 3D culture were also more responsive to caffeine, likely reflecting an increased availability of calcium in the sarcoplasmic reticulum. Taken together, these studies provide novel strategies for maturing iPSC-derived CMs that may have applications in drug screening and transplantation therapies to treat heart disease. PMID:27392582

  12. Human induced pluripotent stem cell-derived fiber-shaped cardiac tissue on a chip.

    PubMed

    Morimoto, Y; Mori, S; Sakai, F; Takeuchi, S

    2016-06-21

    We propose a method for the production of a fiber-shaped three-dimensional (3D) cellular construct of human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) for the quantification of the contractile force. By culturing the cardiomyocytes in a patterned hydrogel structure with fixed edges, we succeeded in fabricating hiPS-CM fibers with aligned cardiomyocytes. The fiber generated contractile force along the fiber direction due to the hiPS-CM alignment, and we were able to measure its contractile force accurately. Furthermore, to demonstrate the drug reactivity of hiPS-CM fibers, the changes in the contractile frequency and force following treatment with isoproterenol and propranolol were observed. We believe that hiPS-CM fibers will be a useful tool for pharmacokinetic analyses during drug development. PMID:27217209

  13. Prospects for In Vitro Myofilament Maturation in Stem Cell-Derived Cardiac Myocytes

    PubMed Central

    Schwan, Jonas; Campbell, Stuart G

    2015-01-01

    Cardiomyocytes derived from human stem cells are quickly becoming mainstays of cardiac regenerative medicine, in vitro disease modeling, and drug screening. Their suitability for such roles may seem obvious, but assessments of their contractile behavior suggest that they have not achieved a completely mature cardiac muscle phenotype. This could be explained in part by an incomplete transition from fetal to adult myofilament protein isoform expression. In this commentary, we review evidence that supports this hypothesis and discuss prospects for ultimately generating engineered heart tissue specimens that behave similarly to adult human myocardium. We suggest approaches to better characterize myofilament maturation level in these in vitro systems, and illustrate how new computational models could be used to better understand complex relationships between muscle contraction, myofilament protein isoform expression, and maturation. PMID:26085788

  14. An Engineered Cardiac Reporter Cell Line Identifies Human Embryonic Stem Cell-Derived Myocardial Precursors

    PubMed Central

    Mihardja, Shirley S.; Liszewski, Walter; Erle, David J.; Lee, Randall J.; Bernstein, Harold S.

    2011-01-01

    Unlike some organs, the heart is unable to repair itself after injury. Human embryonic stem cells (hESCs) grow and divide indefinitely while maintaining the potential to develop into many tissues of the body. As such, they provide an unprecedented opportunity to treat human diseases characterized by tissue loss. We have identified early myocardial precursors derived from hESCs (hMPs) using an α-myosin heavy chain (αMHC)-GFP reporter line. We have demonstrated by immunocytochemistry and quantitative real-time PCR (qPCR) that reporter activation is restricted to hESC-derived cardiomyocytes (CMs) differentiated in vitro, and that hMPs give rise exclusively to muscle in an in vivo teratoma formation assay. We also demonstrate that the reporter does not interfere with hESC genomic stability. Importantly, we show that hMPs give rise to atrial, ventricular and specialized conduction CM subtypes by qPCR and microelectrode array analysis. Expression profiling of hMPs over the course of differentiation implicate Wnt and transforming growth factor-β signaling pathways in CM development. The identification of hMPs using this αMHC-GFP reporter line will provide important insight into the pathways regulating human myocardial development, and may provide a novel therapeutic reagent for the treatment of cardiac disease. PMID:21245908

  15. Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells in Phenotypic Screening: A Transforming Growth Factor-β Type 1 Receptor Kinase Inhibitor Induces Efficient Cardiac Differentiation.

    PubMed

    Drowley, Lauren; Koonce, Chad; Peel, Samantha; Jonebring, Anna; Plowright, Alleyn T; Kattman, Steven J; Andersson, Henrik; Anson, Blake; Swanson, Bradley J; Wang, Qing-Dong; Brolen, Gabriella

    2016-02-01

    Several progenitor cell populations have been reported to exist in hearts that play a role in cardiac turnover and/or repair. Despite the presence of cardiac stem and progenitor cells within the myocardium, functional repair of the heart after injury is inadequate. Identification of the signaling pathways involved in the expansion and differentiation of cardiac progenitor cells (CPCs) will broaden insight into the fundamental mechanisms playing a role in cardiac homeostasis and disease and might provide strategies for in vivo regenerative therapies. To understand and exploit cardiac ontogeny for drug discovery efforts, we developed an in vitro human induced pluripotent stem cell-derived CPC model system using a highly enriched population of KDR(pos)/CKIT(neg)/NKX2.5(pos) CPCs. Using this model system, these CPCs were capable of generating highly enriched cultures of cardiomyocytes under directed differentiation conditions. In order to facilitate the identification of pathways and targets involved in proliferation and differentiation of resident CPCs, we developed phenotypic screening assays. Screening paradigms for therapeutic applications require a robust, scalable, and consistent methodology. In the present study, we have demonstrated the suitability of these cells for medium to high-throughput screens to assess both proliferation and multilineage differentiation. Using this CPC model system and a small directed compound set, we identified activin-like kinase 5 (transforming growth factor-β type 1 receptor kinase) inhibitors as novel and potent inducers of human CPC differentiation to cardiomyocytes. Significance: Cardiac disease is a leading cause of morbidity and mortality, with no treatment available that can result in functional repair. This study demonstrates how differentiation of induced pluripotent stem cells can be used to identify and isolate cell populations of interest that can translate to the adult human heart. Two separate examples of phenotypic

  16. Coupling primary and stem cell-derived cardiomyocytes in an in vitro model of cardiac cell therapy.

    PubMed

    Aratyn-Schaus, Yvonne; Pasqualini, Francesco S; Yuan, Hongyan; McCain, Megan L; Ye, George J C; Sheehy, Sean P; Campbell, Patrick H; Parker, Kevin Kit

    2016-02-15

    The efficacy of cardiac cell therapy depends on the integration of existing and newly formed cardiomyocytes. Here, we developed a minimal in vitro model of this interface by engineering two cell microtissues (μtissues) containing mouse cardiomyocytes, representing spared myocardium after injury, and cardiomyocytes generated from embryonic and induced pluripotent stem cells, to model newly formed cells. We demonstrated that weaker stem cell-derived myocytes coupled with stronger myocytes to support synchronous contraction, but this arrangement required focal adhesion-like structures near the cell-cell junction that degrade force transmission between cells. Moreover, we developed a computational model of μtissue mechanics to demonstrate that a reduction in isometric tension is sufficient to impair force transmission across the cell-cell boundary. Together, our in vitro and in silico results suggest that mechanotransductive mechanisms may contribute to the modest functional benefits observed in cell-therapy studies by regulating the amount of contractile force effectively transmitted at the junction between newly formed and spared myocytes. PMID:26858266

  17. Human embryonic stem cell-derived cardiomyocytes engraft but do not alter cardiac remodeling after chronic infarction in rats

    PubMed Central

    Fernandes, S; Naumova, AV; Zhu, WZ; Laflamme, MA; Gold, J; Murry, CE

    2010-01-01

    Background Previous studies indicated that, in an acute myocardial infarction model, human embryonic stem cell-derived cardiomyocytes (hESC-CM) injected with a pro-survival cocktail (PSC) can preserve contractile function. Because patients with established heart failure may also benefit from cell transplantation, we evaluated the physiological effects of hESC-CM transplanted into a chronic model of myocardial infarction. Methods and Results Intramyocardial injection of hESC-CM with PSC was performed in nude rats at 1 month following ischemia-reperfusion. The left ventricular function of hESC-CM injected rats was evaluated at 1, 2 and 3 months after the cell injection procedure and was compared to 3 control groups (rats injected with serum-free media, PSC-only, or non-cardiac human cells in PSC). Histology at 3 months revealed that human cardiomyocytes survive, develop increased sarcomere organization and are still proliferating. Despite successful engraftment, both echocardiography and MRI analyses showed no significant difference in left ventricular structure or function between these 4 groups at any time point of the study, suggesting that human cardiomyocytes do not affect cardiac remodeling in a rat model of chronic myocardial infarction. Conclusion When injected into a chronic infarct model, hESC-CM can engraft, survive and form grafts with striated cardiomyocytes at least as well as was previously observed in an acute myocardial infarction model. However, although hESC-CM transplantation can attenuate the progression of heart failure in an acute model, the same hESC-CM injection protocol is insufficient to restore heart function or to alter adverse remodeling of a chronic myocardial infarction model. PMID:20854826

  18. A Microfluidic Bioreactor for Toxicity Testing of Stem Cell Derived 3D Cardiac Bodies.

    PubMed

    Christoffersson, Jonas; Bergström, Gunnar; Schwanke, Kristin; Kempf, Henning; Zweigerdt, Robert; Mandenius, Carl-Fredrik

    2016-01-01

    Modeling tissues and organs using conventional 2D cell cultures is problematic as the cells rapidly lose their in vivo phenotype. In microfluidic bioreactors the cells reside in microstructures that are continuously perfused with cell culture medium to provide a dynamic environment mimicking the cells natural habitat. These micro scale bioreactors are sometimes referred to as organs-on-chips and are developed in order to improve and extend cell culture experiments. Here, we describe the two manufacturing techniques photolithography and soft lithography that are used in order to easily produce microfluidic bioreactors. The use of these bioreactors is exemplified by a toxicity assessment on 3D clustered human pluripotent stem cells (hPSC)-derived cardiomyocytes by beating frequency imaging. PMID:27052611

  19. A multi-parameter in vitro screen in human stem cell-derived cardiomyocytes identifies ponatinib-induced structural and functional cardiac toxicity.

    PubMed

    Talbert, Dominique R; Doherty, Kimberly R; Trusk, Patricia B; Moran, Diarmuid M; Shell, Scott A; Bacus, Sarah

    2015-01-01

    Ponatinib, a multi-targeted TKI and potent pan-ABL inhibitor, approved for the treatment of Ph + ALL and CML, was temporarily withdrawn from the U.S. market due to severe vascular adverse events. Cardiac-specific toxicities including myocardial infarction, severe congestive heart failure, and cardiac arrhythmias have also been shown with ponatinib. Targeted oncology agents such as ponatinib have transformed cancer treatment but often induce toxicity due to inhibition of survival pathways shared by both cancer and cardiac cells. These toxicities are often missed by the standard preclinical toxicity assessment methods, which include human Ether-à-go-go-related gene (hERG) and animal toxicity testing. In this study, we show that a multiparameter in vitro toxicity screening approach using human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) accurately predicted the cardiac toxicity potential of ponatinib. This in vitro model evaluated ponatinib's effect on the overall cell health, mitochondrial stress, and function of hiPSC-CM and also provided mechanistic insight into the signaling pathways and cellular structures altered with treatment. We show here that ponatinib rapidly inhibits prosurvival signaling pathways, induces structural cardiac toxicity (as shown by actin cytoskeleton damage, mitochondrial stress, cell death, and troponin secretion), and disrupts cardiac cell beating. Most of these effects occurred at doses between 10× and 50× ponatinib's Cmax, a dose range shown to be relevant for accurate prediction of in vivo toxicity. Together these studies show that a comprehensive in vitro screening tool in a more relevant human cardiac cell model can improve the detection of cardiac toxicity with targeted oncology agents such as ponatinib. PMID:25304212

  20. Drug Discovery Models and Toxicity Testing Using Embryonic and Induced Pluripotent Stem-Cell-Derived Cardiac and Neuronal Cells

    PubMed Central

    Deshmukh, Rahul S.; Kovács, Krisztián A; Dinnyés, András

    2012-01-01

    Development of induced pluripotent stem cells (iPSCs) using forced expression of specific sets of transcription factors has changed the field of stem cell research extensively. Two important limitations for research application of embryonic stem cells (ESCs), namely, ethical and immunological issues, can be circumvented using iPSCs. Since the development of first iPSCs, tremendous effort has been directed to the development of methods to increase the efficiency of the process and to reduce the extent of genomic modifications associated with the reprogramming procedure. The established lineage-specific differentiation protocols developed for ESCs are being applied to iPSCs, as they have great potential in regenerative medicine for cell therapy, disease modeling either for drug development or for fundamental science, and, last but not least, toxicity testing. This paper reviews efforts aimed at practical development of iPSC differentiation to neural/cardiac lineages and further the use of these iPSCs-derived cells for drug development and toxicity testing. PMID:22654918

  1. Evaluation of Changes in Morphology and Function of Human Induced Pluripotent Stem Cell Derived Cardiomyocytes (HiPSC-CMs) Cultured on an Aligned-Nanofiber Cardiac Patch

    PubMed Central

    Khan, Mahmood; Xu, Yanyi; Hua, Serena; Johnson, Jed; Belevych, Andriy; Janssen, Paul M. L.; Gyorke, Sandor; Guan, Jianjun; Angelos, Mark G.

    2015-01-01

    Introduction Dilated cardiomyopathy is a major cause of progressive heart failure. Utilization of stem cell therapy offers a potential means of regenerating viable cardiac tissue. However, a major obstacle to stem cell therapy is the delivery and survival of implanted stem cells in the ischemic heart. To address this issue, we have developed a biomimetic aligned nanofibrous cardiac patch and characterized the alignment and function of human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) cultured on this cardiac patch. This hiPSC-CMs seeded patch was compared with hiPSC-CMs cultured on standard flat cell culture plates. Methods hiPSC-CMs were cultured on; 1) a highly aligned polylactide-co-glycolide (PLGA) nanofiber scaffold (~50 microns thick) and 2) on a standard flat culture plate. Scanning electron microscopy (SEM) was used to determine alignment of PLGA nanofibers and orientation of the cells on the respective surfaces. Analysis of gap junctions (Connexin-43) was performed by confocal imaging in both the groups. Calcium cycling and patch-clamp technique were performed to measure calcium transients and electrical coupling properties of cardiomyocytes. Results SEM demonstrated >90% alignment of the nanofibers in the patch which is similar to the extracellular matrix of decellularized rat myocardium. Confocal imaging of the cardiomyocytes demonstrated symmetrical alignment in the same direction on the aligned nanofiber patch in sharp contrast to the random appearance of cardiomyocytes cultured on a tissue culture plate. The hiPSC-CMs cultured on aligned nanofiber cardiac patches showed more efficient calcium cycling compared with cells cultured on standard flat surface culture plates. Quantification of mRNA with qRT-PCR confirmed that these cardiomyocytes expressed α-actinin, troponin-T and connexin-43 in-vitro. Conclusions Overall, our results demonstrated changes in morphology and function of human induced pluripotent derived cardiomyocytes

  2. Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways

    PubMed Central

    Furniss, Mike; Mussio, Jodie; Davis, Myrtle

    2015-01-01

    There is a need to develop mechanism-based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are gaining rapid acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC-CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathway(s) in hiPSC-CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression, functional integrity of signaling pathway(s) of interest and that characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional and biochemical endpoints to interrogate compound effects on cardiomyocytes. PMID:26331525

  3. Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) to Monitor Compound Effects on Cardiac Myocyte Signaling Pathways.

    PubMed

    Guo, Liang; Eldridge, Sandy; Furniss, Mike; Mussio, Jodie; Davis, Myrtle

    2015-01-01

    There is a need to develop mechanism-based assays to better inform risk of cardiotoxicity. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are rapidly gaining acceptance as a biologically relevant in vitro model for use in drug discovery and cardiotoxicity screens. Utilization of hiPSC-CMs for mechanistic investigations would benefit from confirmation of the expression and activity of cellular pathways that are known to regulate cardiac myocyte viability and function. This unit describes an approach to demonstrate the presence and function of signaling pathways in hiPSC-CMs and the effects of treatments on these pathways. We present a workflow that employs protocols to demonstrate protein expression and functional integrity of signaling pathway(s) of interest and to characterize biological consequences of signaling modulation. These protocols utilize a unique combination of structural, functional, and biochemical endpoints to interrogate compound effects on cardiomyocytes. PMID:26331525

  4. Comparison of Human Embryonic Stem Cell-Derived Cardiomyocytes, Cardiovascular Progenitors, and Bone Marrow Mononuclear Cells for Cardiac Repair

    PubMed Central

    Fernandes, Sarah; Chong, James J.H.; Paige, Sharon L.; Iwata, Mineo; Torok-Storb, Beverly; Keller, Gordon; Reinecke, Hans; Murry, Charles E.

    2015-01-01

    Summary Cardiomyocytes derived from human embryonic stem cells (hESC-CMs) can improve the contractility of injured hearts. We hypothesized that mesodermal cardiovascular progenitors (hESC-CVPs), capable of generating vascular cells in addition to cardiomyocytes, would provide superior repair by contributing to multiple components of myocardium. We performed a head-to-head comparison of hESC-CMs and hESC-CVPs and compared these with the most commonly used clinical cell type, human bone marrow mononuclear cells (hBM-MNCs). In a nude rat model of myocardial infarction, hESC-CMs and hESC-CVPs generated comparable grafts. Both similarly improved systolic function and ventricular dilation. Furthermore, only rare human vessels formed from hESC-CVPs. hBM-MNCs attenuated ventricular dilation and enhanced host vascularization without engrafting long-term or improving contractility. Thus, hESC-CMs and CVPs show similar efficacy for cardiac repair, and both are more efficient than hBM-MNCs. However, hESC-CVPs do not form larger grafts or more significant numbers of human vessels in the infarcted heart. PMID:26607951

  5. Functional Characterization of Human Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Kirsch, Authors Glenn E.; Obejero-Paz, Carlos A.; Bruening-Wright, Andrew

    2014-01-01

    Cardiac toxicity is a leading contributor to late-stage attrition in the drug discovery process and to withdrawal of approved from the market. In vitro assays that enable earlier and more accurate testing for cardiac risk provide early stage predictive indicators that aid in mitigating risk. Human cardiomyocytes, the most relevant subjects for early stage testing, are severely limited in supply. But human stem cell-derived cardiomyocytes (SC-hCM) are readily available from commercial sources and are increasingly used in academic research, drug discovery and safety pharmacology. As a result, SC-hCM electrophysiology has become a valuable tool to assess cardiac risk associated with drugs. This unit describes techniques for recording individual currents carried by sodium, calcium and potassium ions, as well as single cell action potentials, and impedance recordings from contracting syncytia of thousands of interconnected cells. PMID:25152802

  6. Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes

    PubMed Central

    Pilarczyk, Götz; Raulf, Alexandra; Gunkel, Manuel; Fleischmann, Bernd K.; Lemor, Robert; Hausmann, Michael

    2016-01-01

    The present work addresses the question of to what extent a geometrical support acts as a physiological determining template in the setup of artificial cardiac tissue. Surface patterns with alternating concave to convex transitions of cell size dimensions were used to organize and orientate human-induced pluripotent stem cell (hIPSC)-derived cardiac myocytes and mouse neonatal cardiac myocytes. The shape of the cells, as well as the organization of the contractile apparatus recapitulates the anisotropic line pattern geometry being derived from tissue geometry motives. The intracellular organization of the contractile apparatus and the cell coupling via gap junctions of cell assemblies growing in a random or organized pattern were examined. Cell spatial and temporal coordinated excitation and contraction has been compared on plain and patterned substrates. While the α-actinin cytoskeletal organization is comparable to terminally-developed native ventricular tissue, connexin-43 expression does not recapitulate gap junction distribution of heart muscle tissue. However, coordinated contractions could be observed. The results of tissue-like cell ensemble organization open new insights into geometry-dependent cell organization, the cultivation of artificial heart tissue from stem cells and the anisotropy-dependent activity of therapeutic compounds. PMID:26751484

  7. Tissue-Mimicking Geometrical Constraints Stimulate Tissue-Like Constitution and Activity of Mouse Neonatal and Human-Induced Pluripotent Stem Cell-Derived Cardiac Myocytes.

    PubMed

    Pilarczyk, Götz; Raulf, Alexandra; Gunkel, Manuel; Fleischmann, Bernd K; Lemor, Robert; Hausmann, Michael

    2016-01-01

    The present work addresses the question of to what extent a geometrical support acts as a physiological determining template in the setup of artificial cardiac tissue. Surface patterns with alternating concave to convex transitions of cell size dimensions were used to organize and orientate human-induced pluripotent stem cell (hIPSC)-derived cardiac myocytes and mouse neonatal cardiac myocytes. The shape of the cells, as well as the organization of the contractile apparatus recapitulates the anisotropic line pattern geometry being derived from tissue geometry motives. The intracellular organization of the contractile apparatus and the cell coupling via gap junctions of cell assemblies growing in a random or organized pattern were examined. Cell spatial and temporal coordinated excitation and contraction has been compared on plain and patterned substrates. While the α-actinin cytoskeletal organization is comparable to terminally-developed native ventricular tissue, connexin-43 expression does not recapitulate gap junction distribution of heart muscle tissue. However, coordinated contractions could be observed. The results of tissue-like cell ensemble organization open new insights into geometry-dependent cell organization, the cultivation of artificial heart tissue from stem cells and the anisotropy-dependent activity of therapeutic compounds. PMID:26751484

  8. Stem cell derived in vivo-like human cardiac bodies in a microfluidic device for toxicity testing by beating frequency imaging.

    PubMed

    Bergström, Gunnar; Christoffersson, Jonas; Schwanke, Kristin; Zweigerdt, Robert; Mandenius, Carl-Fredrik

    2015-08-01

    Beating in vivo-like human cardiac bodies (CBs) were used in a microfluidic device for testing cardiotoxicity. The CBs, cardiomyocyte cell clusters derived from induced pluripotent stem cells, exhibited typical structural and functional properties of the native human myocardium. The CBs were captured in niches along a perfusion channel in the device. Video imaging was utilized for automatic monitoring of the beating frequency of each individual CB. The device allowed assessment of cardiotoxic effects of drug substances doxorubicin, verapamil and quinidine on the 3D clustered cardiomyocytes. Beating frequency data recorded over a period of 6 hours are presented and compared to literature data. The results indicate that this microfluidic setup with imaging of CB characteristics provides a new opportunity for label-free, non-invasive investigation of toxic effects in a 3D microenvironment. PMID:26135270

  9. Fiber alignment and coculture with fibroblasts improves the differentiated phenotype of murine embryonic stem cell-derived cardiomyocytes for cardiac tissue engineering.

    PubMed

    Parrag, Ian C; Zandstra, Peter W; Woodhouse, Kimberly A

    2012-03-01

    Embryonic stem cells (ESCs) are an important source of cardiomyocytes for regenerating injured myocardium. The successful use of ESC-derived cardiomyocytes in cardiac tissue engineering requires an understanding of the important scaffold properties and culture conditions to promote cell attachment, differentiation, organization, and contractile function. The goal of this work was to investigate how scaffold architecture and coculture with fibroblasts influences the differentiated phenotype of murine ESC-derived cardiomyocytes (mESCDCs). Electrospinning was used to process an elastomeric biodegradable polyurethane (PU) into aligned or unaligned fibrous scaffolds. Bioreactor produced mESCDCs were seeded onto the PU scaffolds either on their own or after pre-seeding the scaffolds with mouse embryonic fibroblasts (MEFs). Viable mESCDCs attached to the PU scaffolds and were functionally contractile in all conditions tested. Importantly, the aligned scaffolds led to the anisotropic organization of rod-shaped cells, improved sarcomere organization, and increased mESCDC aspect ratio (length-to-diameter ratio) when compared to cells on the unaligned scaffolds. In addition, pre-seeding the scaffolds with MEFs improved mESCDC sarcomere formation compared to mESCDCs cultured alone. These results suggest that both fiber alignment and pre-treatment of scaffolds with fibroblasts improve the differentiation of mESCDCs and are important parameters for developing engineered myocardial tissue constructs using ESC-derived cardiac cells. PMID:22006660

  10. Cardiac repair in a porcine model of acute myocardial infarction with human induced pluripotent stem cell-derived cardiovascular cell populations

    PubMed Central

    Ye, Lei; Chang, Ying-Hua; Xiong, Qiang; Zhang, Pengyuan; Zhang, Liying; Somasundaram, Porur; Lepley, Mike; Swingen, Cory; Su, Liping; Wendel, Jacqueline S.; Guo, Jing; Jang, Albert; Rosenbush, Daniel; Greder, Lucas; Dutton, James R.; Zhang, Jianhua; Kamp, Timothy J.; Kaufman, Dan S.; Ge, Ying; Zhang, Jianyi

    2014-01-01

    Summary Human induced pluripotent stem cells (hiPSCs) hold promise for myocardial repair following injury, but preclinical studies in large animal models are required to determine optimal cell preparation and delivery strategies to maximize functional benefits and to evaluate safety. Here, we utilized a porcine model of acute myocardial infarction (MI) to investigate the functional impact of intramyocardial transplantation of hiPSC-derived cardiomyocytes, endothelial cells, and smooth muscle cells, in combination with a 3D fibrin patch loaded with insulin growth factor (IGF)-encapsulated microspheres. hiPSC-derived cardiomyocytes integrated into host myocardium and generated organized sarcomeric structures, and endothelial and smooth muscle cells contributed to host vasculature. Tri-lineage cell transplantation significantly improved left ventricular function, myocardial metabolism, and arteriole density, while reducing infarct size, ventricular wall stress and apoptosis without inducing ventricular arrhythmias. These findings in a large animal MI model highlight the potential of utilizing hiPSC-derived cells for cardiac repair. PMID:25479750

  11. High Throughput Measurement of Ca++ Dynamics in Human Stem Cell-Derived Cardiomyocytes by Kinetic Image Cytometery: A Cardiac Risk Assessment Characterization Using a Large Panel of Cardioactive and Inactive Compounds.

    PubMed

    Lu, Hua Rong; Whittaker, Ross; Price, Jeffrey H; Vega, Raquel; Pfeiffer, Emily R; Cerignoli, Fabio; Towart, Rob; Gallacher, David J

    2015-12-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are emerging as a powerful in vitro model for cardiac safety assessment which may allow for better identification of compounds with poor arrhythmogenic liability profiles early in the drug discovery process. Here, we describe our examination of the Kinetic Image Cytometer (KIC) system's ability to predict adverse compound effects using hiPS-CMs and a library of 53 compounds, the majority of which are known to be cardioactive compounds, and several negative controls. The KIC provides a high throughput method for analyzing intracellular calcium transients. In the cardiomyocyte, intracellular calcium transients integrate the electrochemical signals of the action potential (AP) with the molecular signaling pathways regulating contraction. Drug-induced alterations in the shape and duration of AP result in changes to the shape and duration of the intracellular calcium transient. By examining calcium transient dynamics in hiPS-CMs, KIC can be used as a phenotypic screen to assess compound effects across multiple ion channel types (MITs), detecting MITs, calcium handling and signaling effects. The results of this blinded study indicate that using hiPS-CMs, KIC is able to accurately detect drug-induced changes in Ca(2+) transient dynamics (ie, duration and beat rate) and therefore, may be useful in predicting drug-induced arrhythmogenic liabilities in early de-risking within the drug discovery phase. PMID:26358003

  12. Structural Phenotyping of Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Pasqualini, Francesco Silvio; Sheehy, Sean Paul; Agarwal, Ashutosh; Aratyn-Schaus, Yvonne; Parker, Kevin Kit

    2015-01-01

    Summary Structural phenotyping based on classical image feature detection has been adopted to elucidate the molecular mechanisms behind genetically or pharmacologically induced changes in cell morphology. Here, we developed a set of 11 metrics to capture the increasing sarcomere organization that occurs intracellularly during striated muscle cell development. To test our metrics, we analyzed the localization of the contractile protein α-actinin in a variety of primary and stem-cell derived cardiomyocytes. Further, we combined these metrics with data mining algorithms to unbiasedly score the phenotypic maturity of human-induced pluripotent stem cell-derived cardiomyocytes. PMID:25733020

  13. Stem Cell-Derived Extracellular Vesicles and Immune-Modulation.

    PubMed

    Burrello, Jacopo; Monticone, Silvia; Gai, Chiara; Gomez, Yonathan; Kholia, Sharad; Camussi, Giovanni

    2016-01-01

    Extra-cellular vesicles (EVs) are bilayer membrane structures enriched with proteins, nucleic acids, and other active molecules and have been implicated in many physiological and pathological processes over the past decade. Recently, evidence suggests EVs to play a more dichotomic role in the regulation of the immune system, whereby an immune response may be enhanced or supressed by EVs depending on their cell of origin and its functional state. EVs derived from antigen (Ag)-presenting cells for instance, have been involved in both innate and acquired (or adaptive) immune responses, as Ag carriers or presenters, or as vehicles for delivering active signaling molecules. On the other hand, tumor and stem cell derived EVs have been identified to exert an inhibitory effect on immune responses by carrying immuno-modulatory effectors, such as transcriptional factors, non-coding RNA (Species), and cytokines. In addition, stem cell-derived EVs have also been reported to impair dendritic cell maturation and to regulate the activation, differentiation, and proliferation of B cells. They have been shown to control natural killer cell activity and to suppress the innate immune response (IIR). Studies reporting the role of EVs on T lymphocyte modulation are controversial. Discrepancy in literature may be due to stem cell culture conditions, methods of EV purification, EV molecular content, and functional state of both parental and target cells. However, mesenchymal stem cell-derived EVs were shown to play a more suppressive role by shifting T cells from an activated to a T regulatory phenotype. In this review, we will discuss how stem cell-derived EVs may contribute toward the modulation of the immune response. Collectively, stem cell-derived EVs mainly exhibit an inhibitory effect on the immune system. PMID:27597941

  14. Stem Cell-Derived Extracellular Vesicles and Immune-Modulation

    PubMed Central

    Burrello, Jacopo; Monticone, Silvia; Gai, Chiara; Gomez, Yonathan; Kholia, Sharad; Camussi, Giovanni

    2016-01-01

    Extra-cellular vesicles (EVs) are bilayer membrane structures enriched with proteins, nucleic acids, and other active molecules and have been implicated in many physiological and pathological processes over the past decade. Recently, evidence suggests EVs to play a more dichotomic role in the regulation of the immune system, whereby an immune response may be enhanced or supressed by EVs depending on their cell of origin and its functional state. EVs derived from antigen (Ag)-presenting cells for instance, have been involved in both innate and acquired (or adaptive) immune responses, as Ag carriers or presenters, or as vehicles for delivering active signaling molecules. On the other hand, tumor and stem cell derived EVs have been identified to exert an inhibitory effect on immune responses by carrying immuno-modulatory effectors, such as transcriptional factors, non-coding RNA (Species), and cytokines. In addition, stem cell-derived EVs have also been reported to impair dendritic cell maturation and to regulate the activation, differentiation, and proliferation of B cells. They have been shown to control natural killer cell activity and to suppress the innate immune response (IIR). Studies reporting the role of EVs on T lymphocyte modulation are controversial. Discrepancy in literature may be due to stem cell culture conditions, methods of EV purification, EV molecular content, and functional state of both parental and target cells. However, mesenchymal stem cell-derived EVs were shown to play a more suppressive role by shifting T cells from an activated to a T regulatory phenotype. In this review, we will discuss how stem cell-derived EVs may contribute toward the modulation of the immune response. Collectively, stem cell-derived EVs mainly exhibit an inhibitory effect on the immune system. PMID:27597941

  15. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes

    PubMed Central

    Datta, Rupsa; Heylman, Christopher; George, Steven C.; Gratton, Enrico

    2016-01-01

    In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy. PMID:27231614

  16. Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes.

    PubMed

    Datta, Rupsa; Heylman, Christopher; George, Steven C; Gratton, Enrico

    2016-05-01

    In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy. PMID:27231614

  17. Micropost arrays for measuring stem cell-derived cardiomyocyte contractility.

    PubMed

    Beussman, Kevin M; Rodriguez, Marita L; Leonard, Andrea; Taparia, Nikita; Thompson, Curtis R; Sniadecki, Nathan J

    2016-02-01

    Stem cell-derived cardiomyocytes have the potential to be used to study heart disease and maturation, screen drug treatments, and restore heart function. Here, we discuss the procedures involved in using micropost arrays to measure the contractile forces generated by stem cell-derived cardiomyocytes. Cardiomyocyte contractility is needed for the heart to pump blood, so measuring the contractile forces of cardiomyocytes is a straightforward way to assess their function. Microfabrication and soft lithography techniques are utilized to create identical arrays of flexible, silicone microposts from a common master. Micropost arrays are functionalized with extracellular matrix protein to allow cardiomyocytes to adhere to the tips of the microposts. Live imaging is used to capture videos of the deflection of microposts caused by the contraction of the cardiomyocytes. Image analysis code provides an accurate means to quantify these deflections. The contractile forces produced by a beating cardiomyocyte are calculated by modeling the microposts as cantilever beams. We have used this assay to assess techniques for improving the maturation and contractile function of stem cell-derived cardiomyocytes. PMID:26344757

  18. Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing.

    PubMed

    Pesl, Martin; Pribyl, Jan; Acimovic, Ivana; Vilotic, Aleksandra; Jelinkova, Sarka; Salykin, Anton; Lacampagne, Alain; Dvorak, Petr; Meli, Albano C; Skladal, Petr; Rotrekl, Vladimir

    2016-11-15

    Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in many heart pathologies. Biosensing of these parameters represents an important tool in drug development and disease modeling. Human embryonic stem cells and especially patient specific induced pluripotent stem cell-derived cardiomyocytes are well established as cardiac disease model.. Here, a live stem cell derived embryoid body (EB) based cardiac cell syncytium served as a biorecognition element coupled to the microcantilever probe from atomic force microscope thus providing reliable micromechanical cellular biosensor suitable for whole-day testing. The biosensor was optimized regarding the type of cantilever, temperature and exchange of media; in combination with standardized protocol, it allowed testing of compounds and conditions affecting the biomechanical properties of EB. The studied effectors included calcium , drugs modulating the catecholaminergic fight-or-flight stress response such as the beta-adrenergic blocker metoprolol and the beta-adrenergic agonist isoproterenol. Arrhythmogenic effects were studied using caffeine. Furthermore, with EBs originating from patient's stem cells, this biosensor can help to characterize heart diseases such as dystrophies. PMID:27266660

  19. Stem Cells and Stem Cell-derived Tissues and Their Use in Safety Assessment*

    PubMed Central

    Kolaja, Kyle

    2014-01-01

    Toxicology has long relied on animal models in a tedious approach to understanding risk of exposure to an uncharacterized molecule. Stem cell-derived tissues can be made in high purity, quality, and quantity to enable a new approach to this problem. Currently, stem cell-derived tissues are primarily “generic” genetic backgrounds; the future will see the integration of various genetic backgrounds and complex three-dimensional models to create truly unique in vitro organoids. This minireview focuses on the state of the art of a number of stem cell-derived tissues and details their application in toxicology. PMID:24362027

  20. Methods for Assessing the Electromechanical Integration of Human Pluripotent Stem Cell-Derived Cardiomyocyte Grafts

    PubMed Central

    Zhu, Wei-Zhong; Filice, Dominic; Palpant, Nathan J.; Laflamme, Michael A.

    2014-01-01

    Cardiomyocytes derived from human pluripotent stem cells show tremendous promise for the replacement of myocardium and contractile function lost to infarction. However, until recently, no methods were available to directly determine whether these stem cell-derived grafts actually couple with host myocardium and fire synchronously following transplantation in either intact or injured hearts. To resolve this uncertainty, our group has developed techniques for the intravital imaging of hearts engrafted with stem cell-derived cardiomyocytes that have been modified to express the genetically encoded protein calcium sensor, GCaMP. When combined with the simultaneously recorded electrocardiogram, this protocol allows one to make quantitative assessments as to the presence and extent of host–graft electrical coupling as well as the timing and pattern of graft activation. As described here, this system has been employed to investigate the electromechanical integration of human embryonic stem cell-derived cardiomyocytes in a guinea pig model of cardiac injury, but analogous approaches should be applicable to other human graft cell types and animal models. PMID:25070341

  1. Rapid Cellular Phenotyping of Human Pluripotent Stem Cell-Derived Cardiomyocytes using a Genetically Encoded Fluorescent Voltage Sensor

    PubMed Central

    Leyton-Mange, Jordan S.; Mills, Robert W.; Macri, Vincenzo S.; Jang, Min Young; Butte, Faraz N.; Ellinor, Patrick T.; Milan, David J.

    2014-01-01

    Summary In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity. PMID:24527390

  2. Rapid cellular phenotyping of human pluripotent stem cell-derived cardiomyocytes using a genetically encoded fluorescent voltage sensor.

    PubMed

    Leyton-Mange, Jordan S; Mills, Robert W; Macri, Vincenzo S; Jang, Min Young; Butte, Faraz N; Ellinor, Patrick T; Milan, David J

    2014-02-11

    In addition to their promise in regenerative medicine, pluripotent stem cells have proved to be faithful models of many human diseases. In particular, patient-specific stem cell-derived cardiomyocytes recapitulate key features of several life-threatening cardiac arrhythmia syndromes. For both modeling and regenerative approaches, phenotyping of stem cell-derived tissues is critical. Cellular phenotyping has largely relied upon expression of lineage markers rather than physiologic attributes. This is especially true for cardiomyocytes, in part because electrophysiological recordings are labor intensive. Likewise, most optical voltage indicators suffer from phototoxicity, which damages cells and degrades signal quality. Here we present the use of a genetically encoded fluorescent voltage indicator, ArcLight, which we demonstrate can faithfully report transmembrane potentials in human stem cell-derived cardiomyocytes. We demonstrate the application of this fluorescent sensor in high-throughput, serial phenotyping of differentiating cardiomyocyte populations and in screening for drug-induced cardiotoxicity. PMID:24527390

  3. The case for induced pluripotent stem cell-derived cardiomyocytes in pharmacological screening

    PubMed Central

    Khan, Jaffar M; Lyon, Alexander R; Harding, Sian E

    2013-01-01

    The current drug screening models are deficient, particularly in detecting cardiac side effects. Human stem cell-derived cardiomyocytes could aid both early cardiotoxicity detection and novel drug discovery. Work over the last decade has generated human embryonic stem cells as potentially accurate sources of human cardiomyocytes, but ethical constraints and poor efficacy in establishing cell lines limit their use. Induced pluripotent stem cells do not require the use of human embryos and have the added advantage of producing patient-specific cardiomyocytes, allowing both generic and disease- and patient-specific pharmacological screening, as well as drug development through disease modelling. A critical question is whether sufficient standards have been achieved in the reliable and reproducible generation of ‘adult-like’ cardiomyocytes from human fibroblast tissue to progress from validation to safe use in practice and drug discovery. This review will highlight the need for a new experimental system, assess the validity of human induced pluripotent stem cell-derived cardiomyocytes and explore what the future may hold for their use in pharmacology. LINKED ARTICLES This article is part of a themed section on Regenerative Medicine and Pharmacology: A Look to the Future. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.169.issue-2 PMID:22845396

  4. Enriched retinal ganglion cells derived from human embryonic stem cells

    PubMed Central

    Gill, Katherine P.; Hung, Sandy S. C.; Sharov, Alexei; Lo, Camden Y.; Needham, Karina; Lidgerwood, Grace E.; Jackson, Stacey; Crombie, Duncan E.; Nayagam, Bryony A.; Cook, Anthony L.; Hewitt, Alex W.; Pébay, Alice; Wong, Raymond C. B.

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  5. Enriched retinal ganglion cells derived from human embryonic stem cells.

    PubMed

    Gill, Katherine P; Hung, Sandy S C; Sharov, Alexei; Lo, Camden Y; Needham, Karina; Lidgerwood, Grace E; Jackson, Stacey; Crombie, Duncan E; Nayagam, Bryony A; Cook, Anthony L; Hewitt, Alex W; Pébay, Alice; Wong, Raymond C B

    2016-01-01

    Optic neuropathies are characterised by a loss of retinal ganglion cells (RGCs) that lead to vision impairment. Development of cell therapy requires a better understanding of the signals that direct stem cells into RGCs. Human embryonic stem cells (hESCs) represent an unlimited cellular source for generation of human RGCs in vitro. In this study, we present a 45-day protocol that utilises magnetic activated cell sorting to generate enriched population of RGCs via stepwise retinal differentiation using hESCs. We performed an extensive characterization of these stem cell-derived RGCs by examining the gene and protein expressions of a panel of neural/RGC markers. Furthermore, whole transcriptome analysis demonstrated similarity of the hESC-derived RGCs to human adult RGCs. The enriched hESC-RGCs possess long axons, functional electrophysiological profiles and axonal transport of mitochondria, suggestive of maturity. In summary, this RGC differentiation protocol can generate an enriched population of functional RGCs from hESCs, allowing future studies on disease modeling of optic neuropathies and development of cell therapies. PMID:27506453

  6. The effect of PVDF-TrFE scaffolds on stem cell derived cardiovascular cells.

    PubMed

    Hitscherich, Pamela; Wu, Siliang; Gordan, Richard; Xie, Lai-Hua; Arinzeh, Treena; Lee, Eun Jung

    2016-07-01

    Recently, electrospun polyvinylidene fluoride (PVDF) and polyvinylidene fluoride-trifluoroethylene (PVDF-TrFE) scaffolds have been developed for tissue engineering applications. These materials have piezoelectric activity, wherein they can generate electric charge with minute mechanical deformations. Since the myocardium is an electroactive tissue, the unique feature of a piezoelectric scaffold is attractive for cardiovascular tissue engineering applications. In this study, we examined the cytocompatibility and function of pluripotent stem cell derived cardiovascular cells including mouse embryonic stem cell-derived cardiomyocytes (mES-CM) and endothelial cells (mES-EC) on PVDF-TrFE scaffolds. MES-CM and mES-EC adhered well to PVDF-TrFE and became highly aligned along the fibers. When cultured on scaffolds, mES-CM spontaneously contracted, exhibited well-registered sarcomeres and expressed classic cardiac specific markers such as myosin heavy chain, cardiac troponin T, and connexin43. Moreover, mES-CM cultured on PVDF-TrFE scaffolds responded to exogenous electrical pacing and exhibited intracellular calcium handling behavior similar to that of mES-CM cultured in 2D. Similar to cardiomyocytes, mES-EC also demonstrated high viability and maintained a mature phenotype through uptake of low-density lipoprotein and expression of classic endothelial cell markers including platelet endothelial cell adhesion molecule, endothelial nitric oxide synthase, and the arterial specific marker, Notch-1. This study demonstrates the feasibility of PVDF-TrFE scaffold as a candidate material for developing engineered cardiovascular tissues utilizing stem cell-derived cells. Biotechnol. Bioeng. 2016;113: 1577-1585. © 2015 Wiley Periodicals, Inc. PMID:26705272

  7. Stem cell-derived systems in toxicology assessment.

    PubMed

    Suter-Dick, Laura; Alves, Paula M; Blaauboer, Bas J; Bremm, Klaus-Dieter; Brito, Catarina; Coecke, Sandra; Flick, Burkhard; Fowler, Paul; Hescheler, Jürgen; Ingelman-Sundberg, Magnus; Jennings, Paul; Kelm, Jens M; Manou, Irene; Mistry, Pratibha; Moretto, Angelo; Roth, Adrian; Stedman, Donald; van de Water, Bob; Beilmann, Mario

    2015-06-01

    Industrial sectors perform toxicological assessments of their potential products to ensure human safety and to fulfill regulatory requirements. These assessments often involve animal testing, but ethical, cost, and time concerns, together with a ban on it in specific sectors, make appropriate in vitro systems indispensable in toxicology. In this study, we summarize the outcome of an EPAA (European Partnership of Alternatives to Animal Testing)-organized workshop on the use of stem cell-derived (SCD) systems in toxicology, with a focus on industrial applications. SCD systems, in particular, induced pluripotent stem cell-derived, provide physiological cell culture systems of easy access and amenable to a variety of assays. They also present the opportunity to apply the vast repository of existing nonclinical data for the understanding of in vitro to in vivo translation. SCD systems from several toxicologically relevant tissues exist; they generally recapitulate many aspects of physiology and respond to toxicological and pharmacological interventions. However, focused research is necessary to accelerate implementation of SCD systems in an industrial setting and subsequent use of such systems by regulatory authorities. Research is required into the phenotypic characterization of the systems, since methods and protocols for generating terminally differentiated SCD cells are still lacking. Organotypical 3D culture systems in bioreactors and microscale tissue engineering technologies should be fostered, as they promote and maintain differentiation and support coculture systems. They need further development and validation for their successful implementation in toxicity testing in industry. Analytical measures also need to be implemented to enable compound exposure and metabolism measurements for in vitro to in vivo extrapolation. The future of SCD toxicological tests will combine advanced cell culture technologies and biokinetic measurements to support regulatory and

  8. Tracing Synaptic Connectivity onto Embryonic Stem Cell-Derived Neurons

    PubMed Central

    Garcia, Isabella; Huang, Longwen; Ung, Kevin; Arenkiel, Benjamin R.

    2012-01-01

    Transsynaptic circuit tracing using genetically modified Rabies virus (RV) is an emerging technology for identifying synaptic connections between neurons. Complementing this methodology, it has become possible to assay the basic molecular and cellular properties of neuronal lineages derived from embryonic stem (ES) cells in vitro, and these properties are under intense investigation towards devising cell replacement therapies. Here, we report the generation of a novel mouse ES cell (mESC) line that harbors the genetic elements to allow RV-mediated transsynaptic circuit tracing in ES cell-derived neurons and their synaptic networks. To facilitate transsynaptic tracing, we have engineered a new reporter allele by introducing cDNA encoding tdTomato, the Rabies-G glycoprotein, and the avian TVA receptor into the ROSA26 locus by gene targeting. We demonstrate high-efficiency differentiation of these novel mESCs into functional neurons, show their capacity to functionally connect with primary neuronal cultures as evidenced by immunohistochemistry and electrophysiological recordings, and show their ability to act as source cells for presynaptic tracing of neuronal networks in vitro and in vivo. Together, our data highlight the potential for using genetically engineered stem cells to investigate fundamental mechanisms of synapse and circuit formation with unambiguous identification of presynaptic inputs onto neuronal populations of interest. PMID:22996827

  9. Trophoblast lineage cells derived from human induced pluripotent stem cells

    SciTech Connect

    Chen, Ying; Wang, Kai; Chandramouli, Gadisetti V.R.; Knott, Jason G.; Leach, Richard

    2013-07-12

    Highlights: •Epithelial-like phenotype of trophoblast lineage cells derived from human iPS cells. •Trophoblast lineage cells derived from human iPS cells exhibit trophoblast function. •Trophoblasts from iPS cells provides a proof-of-concept in regenerative medicine. -- Abstract: Background: During implantation, the blastocyst trophectoderm attaches to the endometrial epithelium and continues to differentiate into all trophoblast subtypes, which are the major components of a placenta. Aberrant trophoblast proliferation and differentiation are associated with placental diseases. However, due to ethical and practical issues, there is almost no available cell or tissue source to study the molecular mechanism of human trophoblast differentiation, which further becomes a barrier to the study of the pathogenesis of trophoblast-associated diseases of pregnancy. In this study, our goal was to generate a proof-of-concept model for deriving trophoblast lineage cells from induced pluripotency stem (iPS) cells from human fibroblasts. In future studies the generation of trophoblast lineage cells from iPS cells established from patient’s placenta will be extremely useful for studying the pathogenesis of individual trophoblast-associated diseases and for drug testing. Methods and results: Combining iPS cell technology with BMP4 induction, we derived trophoblast lineage cells from human iPS cells. The gene expression profile of these trophoblast lineage cells was distinct from fibroblasts and iPS cells. These cells expressed markers of human trophoblasts. Furthermore, when these cells were differentiated they exhibited invasive capacity and placental hormone secretive capacity, suggesting extravillous trophoblasts and syncytiotrophoblasts. Conclusion: Trophoblast lineage cells can be successfully derived from human iPS cells, which provide a proof-of-concept tool to recapitulate pathogenesis of patient placental trophoblasts in vitro.

  10. Islet Endothelial Cells Derived From Mouse Embryonic Stem Cells.

    PubMed

    Jain, Neha; Lee, Eun Jung

    2016-01-01

    The islet endothelium comprises a specialized population of islet endothelial cells (IECs) expressing unique markers such as nephrin and α-1 antitrypsin (AAT) that are not found in endothelial cells in surrounding tissues. However, due to difficulties in isolating and maintaining a pure population of these cells, the information on these islet-specific cells is currently very limited. Interestingly, we have identified a large subpopulation of endothelial cells exhibiting IEC phenotype, while deriving insulin-producing cells from mouse embryonic stem cells (mESCs). These cells were identified by the uptake of low-density lipoprotein (LDL) and were successfully isolated and subsequently expanded in endothelial cell culture medium. Further analysis demonstrated that the mouse embryonic stem cell-derived endothelial cells (mESC-ECs) not only express classical endothelial markers, such as platelet endothelial cell adhesion molecule (PECAM1), thrombomodulin, intercellular adhesion molecule-1 (ICAM-1), and endothelial nitric oxide synthase (eNOS) but also IEC-specific markers such as nephrin and AAT. Moreover, mESC-ECs secrete basement membrane proteins such as collagen type IV, laminin, and fibronectin in culture and form tubular networks on a layer of Matrigel, demonstrating angiogenic activity. Further, mESC-ECs not only express eNOS, but also its eNOS expression is glucose dependent, which is another characteristic phenotype of IECs. With the ability to obtain highly purified IECs derived from pluripotent stem cells, it is possible to closely examine the function of these cells and their interaction with pancreatic β-cells during development and maturation in vitro. Further characterization of tissue-specific endothelial cell properties may enhance our ability to formulate new therapeutic angiogenic approaches for diabetes. PMID:25751085

  11. Stem cells and exosomes in cardiac repair.

    PubMed

    Singla, Dinender K

    2016-04-01

    Cardiac diseases currently lead in the number of deaths per year, giving rise an interest in transplanting embryonic and adult stem cells as a means to improve damaged tissue from conditions such as myocardial infarction and coronary artery disease. After testing these cells as a treatment option in both animal and human models, it is believed that these cells improve the damaged tissue primarily through the release of autocrine and paracrine factors. Major concerns such as teratoma formation, immune response, difficulty harvesting cells, and limited cell proliferation and differentiation, hinder the routine use of these cells as a treatment option in the clinic. The advent of stem cell-derived exosomes circumvent those concerns, while still providing the growth factors, miRNA, and additional cell protective factors that aid in repairing and regenerating the damaged tissue. These exosomes have been found to be anti-apoptotic, anti-fibrotic, pro-angiogenic, as well as enhance cardiac differentiation, all of which are key to repairing damaged tissue. As such, stem cell derived exosomes are considered to be a potential new and novel approach in the treatment of various cardiac diseases. PMID:26848944

  12. Induced pluripotent stem cell-derived neural stem cell therapies for spinal cord injury.

    PubMed

    Lee-Kubli, Corinne A; Lu, Paul

    2015-01-01

    The greatest challenge to successful treatment of spinal cord injury is the limited regenerative capacity of the central nervous system and its inability to replace lost neurons and severed axons following injury. Neural stem cell grafts derived from fetal central nervous system tissue or embryonic stem cells have shown therapeutic promise by differentiation into neurons and glia that have the potential to form functional neuronal relays across injured spinal cord segments. However, implementation of fetal-derived or embryonic stem cell-derived neural stem cell therapies for patients with spinal cord injury raises ethical concerns. Induced pluripotent stem cells can be generated from adult somatic cells and differentiated into neural stem cells suitable for therapeutic use, thereby providing an ethical source of implantable cells that can be made in an autologous fashion to avoid problems of immune rejection. This review discusses the therapeutic potential of human induced pluripotent stem cell-derived neural stem cell transplantation for treatment of spinal cord injury, as well as addressing potential mechanisms, future perspectives and challenges. PMID:25788906

  13. Induced pluripotent stem cell-derived cardiomyocytes: boutique science or valuable arrhythmia model?

    PubMed

    Knollmann, Björn C

    2013-03-15

    This article reviews the strengths and limitations of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) as models of cardiac arrhythmias. Specifically, the article attempts to answer the following questions: Which clinical arrhythmias can be modeled by iPSC-CM? How well can iPSC-CM model adult ventricular myocytes? What are the strengths and limitations of published iPSC-CM arrhythmia models? What new mechanistic insight has been gained? What is the evidence that would support using iPSC-CM to personalize antiarrhythmic drug therapy? The review also discusses the pros and cons of using the iPSC-CM technology for modeling specific genetic arrhythmia disorders, such as long QT syndrome, Brugada Syndrome, or Catecholaminergic Polymorphic Ventricular Tachycardia. PMID:23569106

  14. Glial cell derived neurotrophic factor induces spermatogonial stem cell marker genes in chicken mesenchymal stem cells.

    PubMed

    Boozarpour, Sohrab; Matin, Maryam M; Momeni-Moghaddam, Madjid; Dehghani, Hesam; Mahdavi-Shahri, Naser; Sisakhtnezhad, Sajjad; Heirani-Tabasi, Asieh; Irfan-Maqsood, Muhammad; Bahrami, Ahmad Reza

    2016-06-01

    Mesenchymal stem cells (MSCs) are known with the potential of multi-lineage differentiation. Advances in differentiation technology have also resulted in the conversion of MSCs to other kinds of stem cells. MSCs are considered as a suitable source of cells for biotechnology purposes because they are abundant, easily accessible and well characterized cells. Nowadays small molecules are introduced as novel and efficient factors to differentiate stem cells. In this work, we examined the potential of glial cell derived neurotrophic factor (GDNF) for differentiating chicken MSCs toward spermatogonial stem cells. MSCs were isolated and characterized from chicken and cultured under treatment with all-trans retinoic acid (RA) or glial cell derived neurotrophic factor. Expression analysis of specific genes after 7days of RA treatment, as examined by RT-PCR, proved positive for some germ cell markers such as CVH, STRA8, PLZF and some genes involved in spermatogonial stem cell maintenance like BCL6b and c-KIT. On the other hand, GDNF could additionally induce expression of POU5F1, and NANOG as well as other genes which were induced after RA treatment. These data illustrated that GDNF is relatively more effective in diverting chicken MSCs towards Spermatogonial stem cell -like cells in chickens and suggests GDNF as a new agent to obtain transgenic poultry, nevertheless, exploitability of these cells should be verified by more experiments. PMID:27026484

  15. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 undergo the stochastic cardiomyogenic fate and behave like transient amplifying cells

    SciTech Connect

    Yamada, Yoji; Sakurada, Kazuhiro; Takeda, Yukiji; Gojo, Satoshi; Umezawa, Akihiro . E-mail: umezawa@1985.jukuin.keio.ac.jp

    2007-02-15

    Bone marrow-derived stromal cells can give rise to cardiomyocytes as well as adipocytes, osteocytes, and chondrocytes in vitro. The existence of mesenchymal stem cells has been proposed, but it remains unclear if a single-cell-derived stem cell stochastically commits toward a cardiac lineage. By single-cell marking, we performed a follow-up study of individual cells during the differentiation of 9-15c mesenchymal stromal cells derived from bone marrow cells. Three types of cells, i.e., cardiac myoblasts, cardiac progenitors and multipotent stem cells were differentiated from a single cell, implying that cardiomyocytes are generated stochastically from a single-cell-derived stem cell. We also demonstrated that overexpression of Csx/Nkx2.5 and GATA4, precardiac mesodermal transcription factors, enhanced cardiomyogenic differentiation of 9-15c cells, and the frequency of cardiomyogenic differentiation was increased by co-culturing with fetal cardiomyocytes. Single-cell-derived mesenchymal stem cells overexpressing Csx/Nkx2.5 and GATA4 behaved like cardiac transient amplifying cells, and still retained their plasticity in vivo.

  16. Altered calcium handling and increased contraction force in human embryonic stem cell derived cardiomyocytes following short term dexamethasone exposure.

    PubMed

    Kosmidis, Georgios; Bellin, Milena; Ribeiro, Marcelo C; van Meer, Berend; Ward-van Oostwaard, Dorien; Passier, Robert; Tertoolen, Leon G J; Mummery, Christine L; Casini, Simona

    2015-11-27

    One limitation in using human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) for disease modeling and cardiac safety pharmacology is their immature functional phenotype compared with adult cardiomyocytes. Here, we report that treatment of human embryonic stem cell derived cardiomyocytes (hESC-CMs) with dexamethasone, a synthetic glucocorticoid, activated glucocorticoid signaling which in turn improved their calcium handling properties and contractility. L-type calcium current and action potential properties were not affected by dexamethasone but significantly faster calcium decay, increased forces of contraction and sarcomeric lengths, were observed in hESC-CMs after dexamethasone exposure. Activating the glucocorticoid pathway can thus contribute to mediating hPSC-CMs maturation. PMID:26456652

  17. Transcriptional and Functional Profiling of Human Embryonic Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Xie, Xiaoyan; Fu, Ji-Dong; Drukker, Micha; Lee, Andrew; Li, Ronald A.; Gambhir, Sanjiv S.; Weissman, Irving L.; Robbins, Robert C.; Wu, Joseph C.

    2008-01-01

    Human embryonic stem cells (hESCs) can serve as a potentially limitless source of cells that may enable regeneration of diseased tissue and organs. Here we investigate the use of human embryonic stem cell-derived cardiomyocytes (hESC-CMs) in promoting recovery from cardiac ischemia reperfusion injury in a mouse model. Using microarrays, we have described the hESC-CM transcriptome within the spectrum of changes that occur between undifferentiated hESCs and fetal heart cells. The hESC-CMs expressed cardiomyocyte genes at levels similar to those found in 20-week fetal heart cells, making this population a good source of potential replacement cells in vivo. Echocardiographic studies showed significant improvement in heart function by 8 weeks after transplantation. Finally, we demonstrate long-term engraftment of hESC-CMs by using molecular imaging to track cellular localization, survival, and proliferation in vivo. Taken together, global gene expression profiling of hESC differentiation enables a systems-based analysis of the biological processes, networks, and genes that drive hESC fate decisions, and studies such as this will serve as the foundation for future clinical applications of stem cell therapies. PMID:18941512

  18. Lower Oncogenic Potential of Human Mesenchymal Stem Cells Derived from Cord Blood Compared to Induced Pluripotent Stem Cells

    PubMed Central

    Foroutan, T.; Najmi, M.; Kazemi, N.; Hasanlou, M.; Pedram, A.

    2015-01-01

    Background: In regenerative medicine, use of each of the mesenchymal stem cells derived from bone marrow, cord blood, and adipose tissue, has several cons and pros. Mesenchymal stem cells derived from cord blood have been considered the best source for precursor transplantation. Direct reprogramming of a somatic cell into induced pluripotent stem cells by over-expression of 6 transcription factors Oct4, Sox2, Klf4, lin28, Nanog, and c-Myc has great potential for regenerative medicine, eliminating the ethical issues of embryonic stem cells and the rejection problems of using non-autologous cells. Objective: To compare reprogramming and pluripotent markers OCT4, Sox-2, c-Myc, Klf4, Nanog, and lin28 in mesenchymal stem cells derived from cord blood and induced pluripotent stem cells. Methods: We analyzed the expression level of OCT4, Sox-2, c-Myc, Klf4, Nanog and lin28 genes in human mesenchymal stem cells derived from cord blood and induced pluripotent stem cells by cell culture and RT-PCR. Results: The expression level of pluripotent genes OCT4 and Sox-2, Nanog and lin28 in mesenchymal stem cells derived from cord blood were significantly higher than those in induced pluripotent stem cells. In contrast to OCT-4A and Sox-2, Nanog and lin28, the expression level of oncogenic factors c-Myc and Klf4 were significantly higher in induced pluripotent stem cells than in mesenchymal stem cells derived from cord blood. Conclusion: It could be concluded that mesenchymal stem cells derived from human cord blood have lower oncogenic potential compared to induced pluripotent stem cells. PMID:26306155

  19. [Stem cells and cardiac regeneration].

    PubMed

    Perez Millan, Maria Ines; Lorenti, Alicia

    2006-01-01

    Stem cells are defined by virtue of their functional attributes: absence of tissue specific differentitated markers, capable of proliferation, able to self-maintain the population, able to produce a large number of differentiated, functional progeny, able to regenerate the tissue after injury. Cell therapy is an alternative for the treatment of several diseases, like cardiac diseases (cell cardiomyoplasty). A variety of stem cells could be used for cardiac repair: from cardiac and extracardiac sources. Each cell type has its own profile of advantages, limitations, and practicability issues in specific clinical settings. Differentiation of bone marrow stem cells to cardiomyocyte-like cells have been observed under different culture conditions. The presence of resident cardiac stem cell population capable of differentiation into cardiomyocyte or vascular lineage suggests that these cells could be used for cardiac tissue repair, and represent a great promise for clinical application. Stem cells mobilization by cytokines may also offer a strategy for cardiac regeneration. The use of stem cells (embryonic and adult) may hold the key to replacing cells lost in many devastating diseases. This potential benefit is a major focus for stem cell research. PMID:17240634

  20. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes.

    PubMed

    Kijlstra, Jan David; Hu, Dongjian; Mittal, Nikhil; Kausel, Eduardo; van der Meer, Peter; Garakani, Arman; Domian, Ibrahim J

    2015-12-01

    The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening. PMID:26626178

  1. Integrated Analysis of Contractile Kinetics, Force Generation, and Electrical Activity in Single Human Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Kijlstra, Jan David; Hu, Dongjian; Mittal, Nikhil; Kausel, Eduardo; van der Meer, Peter; Garakani, Arman; Domian, Ibrahim J.

    2015-01-01

    Summary The quantitative analysis of cardiomyocyte function is essential for stem cell-based approaches for the in vitro study of human cardiac physiology and pathophysiology. We present a method to comprehensively assess the function of single human pluripotent stem cell-derived cardiomyocyte (hPSC-CMs) through simultaneous quantitative analysis of contraction kinetics, force generation, and electrical activity. We demonstrate that statistical analysis of movies of contracting hPSC-CMs can be used to quantify changes in cellular morphology over time and compute contractile kinetics. Using a biomechanical model that incorporates substrate stiffness, we calculate cardiomyocyte force generation at single-cell resolution and validate this approach with conventional traction force microscopy. The addition of fluorescent calcium indicators or membrane potential dyes allows the simultaneous analysis of contractility and calcium handling or action potential morphology. Accordingly, our approach has the potential for broad application in the study of cardiac disease, drug discovery, and cardiotoxicity screening. PMID:26626178

  2. Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells.

    PubMed

    Galat, Vasiliy; Galat, Yekaterina; Perepitchka, Mariana; Jennings, Lawrence J; Iannaccone, Philip M; Hendrix, Mary J C

    2016-07-15

    Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation. PMID:27193052

  3. Transgene Reactivation in Induced Pluripotent Stem Cell Derivatives and Reversion to Pluripotency of Induced Pluripotent Stem Cell-Derived Mesenchymal Stem Cells

    PubMed Central

    Galat, Yekaterina; Perepitchka, Mariana; Jennings, Lawrence J.; Iannaccone, Philip M.; Hendrix, Mary J.C.

    2016-01-01

    Induced pluripotent stem cells (iPSCs) have enormous potential in regenerative medicine and disease modeling. It is now felt that clinical trials should be performed with iPSCs derived with nonintegrative constructs. Numerous studies, however, including those describing disease models, are still being published using cells derived from iPSCs generated with integrative constructs. Our experimental work presents the first evidence of spontaneous transgene reactivation in vitro in several cellular types. Our results show that the transgenes were predominantly silent in parent iPSCs, but in mesenchymal and endothelial iPSC derivatives, the transgenes experienced random upregulation of Nanog and c-Myc. Additionally, we provide evidence of spontaneous secondary reprogramming and reversion to pluripotency in mesenchymal stem cells derived from iPSCs. These findings strongly suggest that the studies, which use cellular products derived from iPSCs generated with retro- or lentiviruses, should be evaluated with consideration of the possibility of transgene reactivation. The in vitro model described here provides insight into the earliest events of culture transformation and suggests the hypothesis that reversion to pluripotency may be responsible for the development of tumors in cell replacement experiments. The main goal of this work, however, is to communicate the possibility of transgene reactivation in retro- or lenti-iPSC derivatives and the associated loss of cellular fidelity in vitro, which may impact the outcomes of disease modeling and related experimentation. PMID:27193052

  4. Effects of cardioactive drugs on human induced pluripotent stem cell derived long QT syndrome cardiomyocytes.

    PubMed

    Kuusela, Jukka; Kujala, Ville J; Kiviaho, Anna; Ojala, Marisa; Swan, Heikki; Kontula, Kimmo; Aalto-Setälä, Katriina

    2016-01-01

    Human induced pluripotent stem cells (hiPSC) have enabled a major step forward in pathophysiologic studies of inherited diseases and may also prove to be valuable in in vitro drug testing. Long QT syndrome (LQTS), characterized by prolonged cardiac repolarization and risk of sudden death, may be inherited or result from adverse drug effects. Using a microelectrode array platform, we investigated the effects of six different drugs on the electrophysiological characteristics of human embryonic stem cell-derived cardiomyocytes as well as hiPSC-derived cardiomyocytes from control subjects and from patients with type 1 (LQT1) and type 2 (LQT2) of LQTS. At baseline the repolarization time was significantly longer in LQTS cells compared to controls. Isoprenaline increased the beating rate of all cell lines by 10-73 % but did not show any arrhythmic effects in any cell type. Different QT-interval prolonging drugs caused prolongation of cardiac repolarization by 3-13 % (cisapride), 10-20 % (erythromycin), 8-23 % (sotalol), 16-42 % (quinidine) and 12-27 % (E-4031), but we did not find any systematic differences in sensitivity between the control, LQT1 and LQT2 cell lines. Sotalol, quinidine and E-4031 also caused arrhythmic beats and beating arrests in some cases. In summary, the drug effects on these patient-specific cardiomyocytes appear to recapitulate clinical observations and provide further evidence that these cells can be applied for in vitro drug testing to probe their vulnerability to arrhythmia. PMID:27026928

  5. Pharmacoelectrophysiology of viral-free induced pluripotent stem cell-derived human cardiomyocytes.

    PubMed

    Mehta, Ashish; Chung, YingYing; Sequiera, Glen Lester; Wong, Philip; Liew, Reginald; Shim, Winston

    2013-02-01

    Development of pharmaceutical agents for cardiac indication demands elaborate safety screening in which assessing repolarization of cardiac cells remains a critical path in risk evaluations. An efficient platform for evaluating cardiac repolarization in vitro significantly facilitates drug developmental programs. In a proof of principle study, we examined the effect of antiarrhythmogenic drugs (Vaughan Williams class I-IV) and noncardiac active drugs (terfenadine and cisapride) on the repolarization profile of viral-free human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Extracellular field potential (FP) recording using microelectrode arrays demonstrated significant delayed repolarization as prolonged corrected FP durations (cFPDs) by class I (quinidine and flecainide), class III (sotalol and amiodarone), and class IV (verapamil), whereas class II drugs (propranolol and nadolol) had no effects. Consistent with their sodium channel-blocking ability, class I drugs also significantly reduced FPmin and conduction velocity. Although lidocaine (class IB) had no effects on cFPDs, verapamil shortened cFPD and FPmin by 25 and 50%, respectively. Furthermore, verapamil reduced beating frequencies drastically. Importantly, the examined drugs exhibited dose-response curve on prolongation of cFPDs at an effective range that correlated significantly with therapeutic plasma concentrations achieved clinically. Consistent with clinical outcomes, drug-induced arrhythmia of tachycardia and bigeminy-like waveforms by quinidine, flecainide, and sotalol was demonstrated at supraphysiological concentrations. Furthermore, off-target effects of terfenadine and cisapride on cFPD and Na( + ) channel blockage were similarly revealed. These results suggest that hiPSC-CMs may be useful for safety evaluation of cardioactive and noncardiac acting drugs for personalized medicine. PMID:23091167

  6. Use of human stem cell derived cardiomyocytes to examine sunitinib mediated cardiotoxicity and electrophysiological alterations

    SciTech Connect

    Cohen, J.D.; Babiarz, J.E.; Abrams, R.M.; Guo, L.; Kameoka, S.; Chiao, E.; Taunton, J.; Kolaja, K.L.

    2011-11-15

    Sunitinib, an oral tyrosine kinase inhibitor approved to treat advanced renal cell carcinoma and gastrointestinal stroma tumor, is associated with clinical cardiac toxicity. Although the precise mechanism of sunitinib cardiotoxicity is not known, both the key metabolic energy regulator, AMP-activated protein kinase (AMPK), and ribosomal S 6 kinase (RSK) have been hypothesized as causative, albeit based on rodent models. To study the mechanism of sunitinib-mediated cardiotoxicity in a human model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) having electrophysiological and contractile properties of native cardiac tissue were investigated. Sunitinib was cardiotoxic in a dose-dependent manner with an IC{sub 50} in the low micromolar range, observed by a loss of cellular ATP, an increase in oxidized glutathione, and induction of apoptosis in iPSC-CMs. Pretreatment of iPSC-CMs with AMPK activators AICAR or metformin, increased the phosphorylation of pAMPK-T172 and pACC-S79, but only marginally attenuated sunitinib mediated cell death. Furthermore, additional inhibitors of AMPK were not directly cytotoxic to iPSC-CMs up to 250 {mu}M concentrations. Inhibition of RSK with a highly specific, irreversible, small molecule inhibitor (RSK-FMK-MEA) did not induce cytotoxicity in iPSC-CMs below 250 {mu}M. Extensive electrophysiological analysis of sunitinib and RSK-FMK-MEA mediated conduction effects were performed. Taken together, these findings suggest that inhibition of AMPK and RSK are not a major component of sunitinib-induced cardiotoxicity. Although the exact mechanism of cardiotoxicity of sunitinib is not known, it is likely due to inhibition of multiple kinases simultaneously. These data highlight the utility of human iPSC-CMs in investigating the potential molecular mechanisms underlying drug-induced cardiotoxicity. -- Highlights: Black-Right-Pointing-Pointer Cytoxic effect of sunitinib on human stem cell derived cardiomyocytes Black

  7. Induced pluripotent stem cell-derived mesenchymal stem cells: A leap toward personalized therapies.

    PubMed

    Whitt, Jason; Vallabhaneni, Krishna C; Penfornis, Patrice; Pochampally, Radhika

    2016-01-01

    Mesenchymal Stem/stromal cell (MSCs) transplantation procedures have been used since the 1960's to treat leukemia and other diseases, but due to the risks involved only patients with life threatening illnesses were typically subjected to the transplantation procedure until the last decade. Recent advancements in transplantation techniques have made it more feasible to use it for non-life-threatening diseases. However, the potential uses for stem cells are still limited by their rarity, and, in the case of allogeneic transplants, graft-vs.-host complications. An evolving alternative to conventional stem cell therapies is induced pluripotent stem-cell derived mesenchymal stem/stromal cells (iPSC- MSCs), which have a multi-lineage potential comparable to conventionally acquired MSCs with the added benefit of being less immunoreactive. However there are still many hurdles left to be overcome before they can be used regularly for personalized therapies. This review will focus on recent advancements that have been made regarding the role MSCs play in tumor development and the potential uses iPSC-MSCs may have in future cancer treatment. PMID:26423301

  8. Human progenitor cells derived from cardiac adipose tissue ameliorate myocardial infarction in rodents.

    PubMed

    Bayes-Genis, Antoni; Soler-Botija, Carolina; Farré, Jordi; Sepúlveda, Pilar; Raya, Angel; Roura, Santiago; Prat-Vidal, Cristina; Gálvez-Montón, Carolina; Montero, José Anastasio; Büscher, Dirk; Izpisúa Belmonte, Juan Carlos

    2010-11-01

    Myocardial infarction caused by vascular occlusion results in the formation of nonfunctional fibrous tissue. Cumulative evidence indicates that cell therapy modestly improves cardiac function; thus, novel cell sources with the potential to repair injured tissue are actively sought. Here, we identify and characterize a cell population of cardiac adipose tissue-derived progenitor cells (ATDPCs) from biopsies of human adult cardiac adipose tissue. Cardiac ATDPCs express a mesenchymal stem cell-like marker profile (strongly positive for CD105, CD44, CD166, CD29 and CD90) and have immunosuppressive capacity. Moreover, cardiac ATDPCs have an inherent cardiac-like phenotype and were able to express de novo myocardial and endothelial markers in vitro but not to differentiate into adipocytes. In addition, when cardiac ATDPCs were transplanted into injured myocardium in mouse and rat models of myocardial infarction, the engrafted cells expressed cardiac (troponin I, sarcomeric α-actinin) and endothelial (CD31) markers, vascularization increased, and infarct size was reduced in mice and rats. Moreover, significant differences between control and cell-treated groups were found in fractional shortening and ejection fraction, and the anterior wall remained significantly thicker 30days after cardiac delivery of ATDPCs. Finally, cardiac ATDPCs secreted proangiogenic factors under in vitro hypoxic conditions, suggesting a paracrine effect to promote local vascularization. Our results indicate that the population of progenitor cells isolated from human cardiac adipose tissue (cardiac ATDPCs) may be valid candidates for future use in cell therapy to regenerate injured myocardium. PMID:20713059

  9. Lost in translation: pluripotent stem cell-derived hematopoiesis

    PubMed Central

    Ackermann, Mania; Liebhaber, Steffi; Klusmann, Jan-Henning; Lachmann, Nico

    2015-01-01

    Pluripotent stem cells (PSCs) such as embryonic stem cells or induced pluripotent stem cells represent a promising cell type to gain novel insights into human biology. Understanding the differentiation process of PSCs in vitro may allow for the identification of cell extrinsic/intrinsic factors, driving the specification process toward all cell types of the three germ layers, which may be similar to the human in vivo scenario. This would not only lay the ground for an improved understanding of human embryonic development but would also contribute toward the generation of novel cell types used in cell replacement therapies. In this line, especially the developmental process of mesodermal cells toward the hematopoietic lineage is of great interest. Therefore, this review highlights recent progress in the field of hematopoietic specification of pluripotent stem cell sources. In addition, we would like to shed light on emerging factors controlling primitive and definitive hematopoietic development and to highlight recent approaches to improve the differentiation potential of PSC sources toward hematopoietic stem/progenitor cells. While the generation of fully defined hematopoietic stem cells from PSCs remains challenging in vitro, we here underline the instructive role of cell extrinsic factors such as cytokines for the generation of PSC-derived mature hematopoietic cells. Thus, we have comprehensively examined the role of cytokines for the derivation of mature hematopoietic cell types such as macrophages, granulocytes, megakaryocytes, erythrocytes, dendritic cells, and cells of the B- and T-cell lineage. PMID:26174486

  10. A strategy to ensure safety of stem cell-derived retinal pigment epithelium cells.

    PubMed

    Choudhary, Parul; Whiting, Paul John

    2016-01-01

    Cell replacement and regenerative therapy using embryonic stem cell-derived material holds promise for the treatment of several pathologies. However, the safety of this approach is of prime importance given the teratogenic potential of residual stem cells, if present in the differentiated cell product. Using the example of embryonic stem cell-derived retinal pigment epithelium (RPE) for the treatment of age-related macular degeneration, we present a novel strategy for ensuring the absence of stem cells in the RPE population. Based on an unbiased screening approach, we identify and validate the expression of CD59, a cell surface marker expressed on RPE but absent on stem cells. We further demonstrate that flow sorting on the basis of CD59 expression can effectively purify RPE and deplete stem cells, resulting in a population free from stem cell impurity. This purification helps to ensure removal of stem cells and hence increases the safety of cells that may be used for clinical transplantation. This strategy can potentially be applied to other pluripotent stem cell-derived material and help mitigate concerns of using such cells for therapy. PMID:27590276

  11. Osteoplant acts on stem cells derived from peripheral blood

    PubMed Central

    Sollazzo, Vincenzo; Palmieri, Annalisa; Girardi, Ambra; Zollino, Ilaria; Brunelli, Giorgio; Spinelli, Giuseppe; Carinci, Francesco

    2010-01-01

    Objectives: The osteoplant is an equine, flexible, heterologous, deantigenic, cortical, and spongy bone tissue, totally reabsorbable, used for implantation of bone tissue, to restore skeletal, even weight-bearing structures. However, how the osteoplant alters osteoblast activity to promote bone formation is poorly understood. Materials and Methods: To study how the osteoplant induces osteoblast differentiation in mesenchymal stem cells, the expression levels of bone-related genes, and mesenchymal stem cell markers are analyzed, using real time Reverse Transcription-Polymerase Chain Reaction (RT-PCR). Results: The osteoplant causes induction of osteoblast transcriptional factors such as osterix (RUNX2), and of bone-related genes such as osteopontin (SPP1) and osteocalcin (BGLAP). In contrast the expression of ENG (CD105) is significantly decreased in stem cells treated with osteoplant, with respect to untreated cells, indicating the differentiation effect of this biomaterial on stem cells. Conclusion: The obtained results can be relevant to better understand the molecular mechanism of bone regeneration and as a model for comparing other materials with similar clinical effects. PMID:20922073

  12. Exposure to Phthalates Affects Calcium Handling and Intercellular Connectivity of Human Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Posnack, Nikki Gillum; Idrees, Rabia; Ding, Hao; Jaimes III, Rafael; Stybayeva, Gulnaz; Karabekian, Zaruhi; Laflamme, Michael A.; Sarvazyan, Narine

    2015-01-01

    Background The pervasive nature of plastics has raised concerns about the impact of continuous exposure to plastic additives on human health. Of particular concern is the use of phthalates in the production of flexible polyvinyl chloride (PVC) products. Di-2-ethylhexyl-phthalate (DEHP) is a commonly used phthalate ester plasticizer that imparts flexibility and elasticity to PVC products. Recent epidemiological studies have reported correlations between urinary phthalate concentrations and cardiovascular disease, including an increased risk of high blood pressure and coronary risk. Yet, there is little direct evidence linking phthalate exposure to adverse effects in human cells, including cardiomyocytes. Methods and Results The effect of DEHP on calcium handling was examined using monolayers of gCAMP3 human embryonic stem cell-derived cardiomyocytes, which contain an endogenous calcium sensor. Cardiomyocytes were exposed to DEHP (5 – 50 μg/mL), and calcium transients were recorded using a Zeiss confocal imaging system. DEHP exposure (24 – 72 hr) had a negative chronotropic and inotropic effect on cardiomyocytes, increased the minimum threshold voltage required for external pacing, and modified connexin-43 expression. Application of Wy-14,643 (100 μM), an agonist for the peroxisome proliferator-activated receptor alpha, did not replicate DEHP’s effects on calcium transient morphology or spontaneous beating rate. Conclusions Phthalates can affect the normal physiology of human cardiomyocytes, including DEHP elicited perturbations in cardiac calcium handling and intercellular connectivity. Our findings call for additional studies to clarify the extent by which phthalate exposure can alter cardiac function, particularly in vulnerable patient populations who are at risk for high phthalate exposure. PMID:25799571

  13. Mesenchymal Stem Cell-Derived Hepatocytes for Functional Liver Replacement

    PubMed Central

    Christ, Bruno; Stock, Peggy

    2012-01-01

    Mesenchymal stem cells represent an alternate cell source to substitute for primary hepatocytes in hepatocyte transplantation because of their multiple differentiation potential and nearly unlimited availability. They may differentiate into hepatocyte-like cells in vitro and maintain specific hepatocyte functions also after transplantation into the regenerating livers of mice or rats both under injury and non-injury conditions. Depending on the underlying liver disease their mode of action is either to replace the diseased liver tissue or to support liver regeneration through their anti-inflammatory and anti-apoptotic as well as their pro-proliferative action. PMID:22737154

  14. Bridging Functional and Structural Cardiotoxicity Assays Using Human Embryonic Stem Cell-Derived Cardiomyocytes for a More Comprehensive Risk Assessment.

    PubMed

    Clements, Mike; Millar, Val; Williams, Angela S; Kalinka, Sian

    2015-11-01

    More relevant and reliable preclinical cardiotoxicity tests are required to improve drug safety and reduce the cost of drug development. Current in vitro testing strategies predominantly take the form of functional assays to predict the potential for drug-induced ECG abnormalities in vivo. Cardiotoxicity can also be structural in nature, so a full and efficient assessment of cardiac liabilities for new chemical entities should account for both these phenomena. As well as providing a more appropriate nonclinical model for in vitro cardiotoxicity testing, human stem cell-derived cardiomyocytes offer an integrated system to study drug impact on cardiomyocyte structure as well as function. Employing human embryonic stem cell-derived cardiacmyocytes (hESC-CMs) on 3 assay platforms with complementary insights into cardiac biology (multielectrode array assay, electrophysiology; impedance assay, cell movement/beating; and high content analysis assay, subcellular structure) we profiled a panel of 13 drugs with well characterized cardiac liabilities (Amiodarone, Aspirin, Astemizole, Axitinib, AZT, Bepridil, Doxorubicin, E-4031, Mexiletine, Rosiglitazone, Sunitinib, Sibutramine, and Verapamil). Our data show good correlations with previous studies and reported clinical observations. Using multiparameter phenotypic profiling techniques we demonstrate the dynamic relationship that exists between functional and structural toxicity, and the benefits of this more holistic approach to risk assessment. We conclude by showing for the first time how the advent of transparent MEA plate technology enables functional and structural cardiotoxic responses to be recorded from the same cell population. This approach more directly links changes in morphology of the hESC-CMs with recorded electrophysiology signatures, offering even greater insight into the wide range of potential drug impacts on cardiac physiology, with a throughput that is more amenable to early drug discovery. PMID:26259608

  15. Permanently Blocked Stem Cells Derived from Breast Cancer Cell Lines

    PubMed Central

    Sajithlal, Gangadharan B.; Rothermund, Kristi; Zhang, Fang; Dabbs, David J.; Latimer, Jean J.; Grant, Stephen G.; Prochownik, Edward V.

    2016-01-01

    Cancer stem cells (CSCs) are thought to be resistant to standard chemotherapeutic drugs and the inimical conditions of the tumor microenvironment. Obtaining CSCs in sufficient quantities and maintaining their undifferentiated state have been major hurdles to their further characterization and to the identification of new pharmaceuticals that preferentially target these cells. We describe here the tagging of CSC-like populations from four human breast cancer cell lines with green fluorescent protein (GFP) under the control of the Oct3/4 stem cell-specific promoter. As expected, GFP was expressed by the CSC-enriched populations. An unanticipated result, however, was that these cells remained blocked in a CSC-like state and tended to be resistant to chemotherapeutic drugs as well as acidotic and hypoxic conditions. These CSC-like cells possessed several other in vitro attributes of CSCs and were able to reproducibly generate tumors in immuno-compromised mice from as few as 100 cells. Moreover, the tumors derived from these cells were comprised almost exclusively of pure CSCs. The ability of the Oct3/4 promoter to block CSC differentiation underscores its potential general utility for obtaining highly purified CSC populations, although the mechanism by which it does so remains undefined and subject to further study. Nonetheless, such stable cell lines should be extremely valuable tools for studying basic questions pertaining to CSC biology and for the initial identification of novel CSC-specific chemotherapeutic agents, which can then be verified in primary CSCs. PMID:20506227

  16. Endothelial cells derived from human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Levenberg, Shulamit; Golub, Justin S.; Amit, Michal; Itskovitz-Eldor, Joseph; Langer, Robert

    2002-04-01

    Human embryonic stem cells have the potential to differentiate into various cell types and, thus, may be useful as a source of cells for transplantation or tissue engineering. We describe here the differentiation steps of human embryonic stem cells into endothelial cells forming vascular-like structures. The human embryonic-derived endothelial cells were isolated by using platelet endothelial cell-adhesion molecule-1 (PECAM1) antibodies, their behavior was characterized in vitro and in vivo, and their potential in tissue engineering was examined. We show that the isolated embryonic PECAM1+ cells, grown in culture, display characteristics similar to vessel endothelium. The cells express endothelial cell markers in a pattern similar to human umbilical vein endothelial cells, their junctions are correctly organized, and they have high metabolism of acetylated low-density lipoprotein. In addition, the cells are able to differentiate and form tube-like structures when cultured on matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells. With further studies, these cells could provide a source of human endothelial cells that could be beneficial for potential applications such as engineering new blood vessels, endothelial cell transplantation into the heart for myocardial regeneration, and induction of angiogenesis for treatment of regional ischemia.

  17. Mesenchymal Stem Cells Derived from Dental Pulp: A Review

    PubMed Central

    Santiago-Osorio, Edelmiro

    2016-01-01

    The mesenchymal stem cells of dental pulp (DPSCs) were isolated and characterized for the first time more than a decade ago as highly clonogenic cells that were able to generate densely calcified colonies. Now, DPSCs are considered to have potential as stem cell source for orthopedic and oral maxillofacial reconstruction, and it has been suggested that they may have applications beyond the scope of the stomatognathic system. To date, most studies have shown that, regardless of their origin in third molars, incisors, or exfoliated deciduous teeth, DPSCs can generate mineralized tissue, an extracellular matrix and structures type dentin, periodontal ligament, and dental pulp, as well as other structures. Different groups worldwide have designed and evaluated new efficient protocols for the isolation, expansion, and maintenance of clinically safe human DPSCs in sufficient numbers for various therapeutics protocols and have discussed the most appropriate route of administration, the possible contraindications to their clinical use, and the parameters to be considered for monitoring their clinical efficacy and proper biological source. At present, DPSC-based therapy is promising but because most of the available evidence was obtained using nonhuman xenotransplants, it is not a mature technology. PMID:26779263

  18. Mesenchymal Stem Cells Derived from Dental Pulp: A Review.

    PubMed

    Ledesma-Martínez, Edgar; Mendoza-Núñez, Víctor Manuel; Santiago-Osorio, Edelmiro

    2016-01-01

    The mesenchymal stem cells of dental pulp (DPSCs) were isolated and characterized for the first time more than a decade ago as highly clonogenic cells that were able to generate densely calcified colonies. Now, DPSCs are considered to have potential as stem cell source for orthopedic and oral maxillofacial reconstruction, and it has been suggested that they may have applications beyond the scope of the stomatognathic system. To date, most studies have shown that, regardless of their origin in third molars, incisors, or exfoliated deciduous teeth, DPSCs can generate mineralized tissue, an extracellular matrix and structures type dentin, periodontal ligament, and dental pulp, as well as other structures. Different groups worldwide have designed and evaluated new efficient protocols for the isolation, expansion, and maintenance of clinically safe human DPSCs in sufficient numbers for various therapeutics protocols and have discussed the most appropriate route of administration, the possible contraindications to their clinical use, and the parameters to be considered for monitoring their clinical efficacy and proper biological source. At present, DPSC-based therapy is promising but because most of the available evidence was obtained using nonhuman xenotransplants, it is not a mature technology. PMID:26779263

  19. Impairment of mesenchymal stem cells derived from oral leukoplakia

    PubMed Central

    Zhang, Zhihui; Song, Jiangyuan; Han, Ying; Mu, Dongdong; Su, Sha; Ji, Xiaoli; Liu, Hongwei

    2015-01-01

    Oral leukoplakia is one of the common precancerous lesions in oral mucosa. To compare the biological characteristics and regenerative capacities of mesenchymal stem cells (MSCs) from oral leukoplakia (epithelial hyperplasia and dysplasia) and normal oral mucosa, MSCs were isolated by enzyme digestion. Then these cells were identified by the expression of MSC related markers, STRO-1, CD105 and CD90, with the absent for the hematopoietic stem cell marker CD34 by flow cytometric detection. The self-renewal ability of MSCs from oral leukoplakia was enhanced, while the multipotent differentiation was descended, compared with MSCs from normal oral mucosa. Fibrin gel was used as a carrier for MSCs transplanted into immunocompromised mice to detect their regenerative capacity. The regenerative capacities of MSCs from oral leukoplakia became impaired partly. Collagen IV (Col IV) and matrix metalloproteinases-9 (MMP-9) were selected to analyze the potential mechanism for the functional changes of MSCs from oral leukoplakia by immunochemical and western blot analysis. The expression of Col IV was decreased and that of MMP-9 was increased by MSCs with the progression of oral leukoplakia, especially in MSCs from epithelial dysplasia. The imbalance between regenerative and metabolic self-regulatory functions of MSCs from oral leukoplakia may be related to the progression of this premalignant disorder. PMID:26617710

  20. Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

    PubMed Central

    Tachibana, Masahito; Amato, Paula; Sparman, Michelle; Gutierrez, Nuria Marti; Tippner-Hedges, Rebecca; Ma, Hong; Kang, Eunju; Fulati, Alimujiang; Lee, Hyo-Sang; Sritanaudomchai, Hathaitip; Masterson, Keith; Larson, Janine; Eaton, Deborah; Sadler-Fredd, Karen; Battaglia, David; Lee, David; Wu, Diana; Jensen, Jeffrey; Patton, Phillip; Gokhale, Sumita; Stouffer, Richard L.; Wolf, Don; Mitalipov, Shoukhrat

    2013-01-01

    SUMMARY Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state. PMID:23683578

  1. Placenta Mesenchymal Stem Cell Derived Exosomes Confer Plasticity on Fibroblasts.

    PubMed

    Tooi, Masayuki; Komaki, Motohiro; Morioka, Chikako; Honda, Izumi; Iwasaki, Kengo; Yokoyama, Naoki; Ayame, Hirohito; Izumi, Yuichi; Morita, Ikuo

    2016-07-01

    Mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) has been reported to enhance wound healing. Exosomes contain nucleic acids, proteins, and lipids, and function as an intercellular communication vehicle for mediating some paracrine effects. However, the function of MSC-derived exosomes (MSC-exo) remains elusive. In this study, we isolated human placenta MSC (PlaMSC)-derived exosomes (PlaMSC-exo) and examined their function in vitro. PlaMSCs were isolated from human term placenta using enzymatic digestion. PlaMSC-exo were prepared from the conditioned medium of PlaMSC (PlaMSC-CM) by ultracentrifugation. The expression of stemness-related genes, such as OCT4 and NANOG, in normal adult human dermal fibroblasts (NHDF) after incubation with PlaMSC-exo was measured by real-time reverse transcriptase PCR analysis (real-time PCR). The effect of PlaMSC-exo on OCT4 transcription activity was assessed using Oct4-EGFP reporter mice-derived dermal fibroblasts. The stimulating effects of PlaMSC-exo on osteoblastic and adipocyte-differentiation of NHDF were evaluated by alkaline phosphatase (ALP), and Alizarin red S- and oil red O-staining, respectively. The expression of osteoblast- and adipocyte-related genes was also assessed by real-time PCR. The treatment of NHDF with PlaMSC-exo significantly upregulated OCT4 and NANOG mRNA expression. PlaMSC-exo also enhanced OCT4 transcription. The NHDF treated with PlaMSC-exo exhibited osteoblastic and adipocyte-differentiation in osteogenic and adipogenic induction media. PlaMSC-exo increase the expression of OCT4 and NANOG mRNA in fibroblasts. As a result, PlaMSC-exo influence the differentiation competence of fibroblasts to both osteoblastic and adipocyte-differentiation. It shows a new feature of MSCs and the possibility of clinical application of MSC-exo. J. Cell. Biochem. 117: 1658-1670, 2016. © 2015 Wiley Periodicals, Inc. PMID:26640165

  2. Maturation of Stem Cell-Derived Beta-cells Guided by the Expression of Urocortin 3

    PubMed Central

    van der Meulen, Talitha; Huising, Mark O.

    2014-01-01

    Type 1 diabetes (T1D) is a devastating disease precipitated by an autoimmune response directed at the insulin-producing beta-cells of the pancreas for which no cure exists. Stem cell-derived beta-cells show great promise for a cure as they have the potential to supply unlimited numbers of cells that could be derived from a patient's own cells, thus eliminating the need for immunosuppression. Current in vitro protocols for the differentiation of stem cell-derived beta-cells can successfully generate pancreatic endoderm cells. In diabetic rodents, such cells can differentiate further along the beta-cell lineage until they are eventually capable of restoring normoglycemia. While these observations demonstrate that stem cell-derived pancreatic endoderm has the potential to differentiate into mature, glucose-responsive beta-cells, the signals that direct differentiation and maturation from pancreatic endoderm onwards remain poorly understood. In this review, we analyze the sequence of events that culminates in the formation of beta-cells during embryonic development. and summarize how current protocols to generate beta-cells have sought to capitalize on this ontogenic template. We place particular emphasis on the current challenges and opportunities which occur in the later stages of beta-cell differentiation and maturation of transplantable stem cell-derived beta-cells. Another focus is on the question how the use of recently identified maturation markers such as urocortin 3 can be instrumental in guiding these efforts. PMID:25148370

  3. The potential of induced pluripotent stem cell derived hepatocytes.

    PubMed

    Hannoun, Zara; Steichen, Clara; Dianat, Noushin; Weber, Anne; Dubart-Kupperschmitt, Anne

    2016-07-01

    Orthotopic liver transplantation remains the only curative treatment for liver disease. However, the number of patients who die while on the waiting list (15%) has increased in recent years as a result of severe organ shortages; furthermore the incidence of liver disease is increasing worldwide. Clinical trials involving hepatocyte transplantation have provided encouraging results. However, transplanted cell function appears to often decline after several months, necessitating liver transplantation. The precise aetiology of the loss of cell function is not clear, but poor engraftment and immune-mediated loss appear to be important factors. Also, primary human hepatocytes (PHH) are not readily available, de-differentiate, and die rapidly in culture. Hepatocytes are available from other sources, such as tumour-derived human hepatocyte cell lines and immortalised human hepatocyte cell lines or porcine hepatocytes. However, all these cells suffer from various limitations such as reduced or differences in functions or risk of zoonotic infections. Due to their significant potential, one possible inexhaustible source of hepatocytes is through the directed differentiation of human induced pluripotent stem cells (hiPSCs). This review will discuss the potential applications and existing limitations of hiPSC-derived hepatocytes in regenerative medicine, drug screening, in vitro disease modelling and bioartificial livers. PMID:26916529

  4. Mesenchymal stem cell-derived exosomes facilitate nasopharyngeal carcinoma progression

    PubMed Central

    Shi, Si; Zhang, Qicheng; Xia, Yunfei; You, Bo; Shan, Ying; Bao, Lili; Li, Li; You, Yiwen; Gu, Zhifeng

    2016-01-01

    Mesenchymal stem cells (MSCs), which are capable of differentiating into multiple cell types, are reported to exert multiple effects on tumor development. However, the relationship between MSCs and nasopharyngeal carcinoma (NPC) cells remains unclear. Exosomes are small membrane vesicles that can be released by several cell types, including MSCs. Exosomes, which can carry membrane and cytoplasmic constituents, have been described as participants in a novel mechanism of cell-to-cell communication. In the present study, we investigated the mechanisms underlying the interaction between MSCs and NPC cells. The data showed that MSCs secreted 40-100 nm heterogeneous small vesicles, which were defined as exosomes. Incubation of NPC cells with MSC-derived exosomes resulted in the uptake of exosomes by the cells, which promoted their proliferation, migration and tumorigenesis. After an extended treatment duration, the tumor cells showed morphological changes and significant changes in the expression of epithelial-mesenchymal transition (EMT) markers. Moreover, we found that FGF19 was highly expressed in MSC-exosomes and that exosomes stimulated NPC progression by activating the FGF19-FGFR4-dependent ERK signaling cascade and by modulating the EMT. All of these data indicated that exosomes participate in a novel mechanism by which MSCs influence NPC progression. PMID:27186416

  5. Cardiac progenitor cell-derived exosomes prevent cardiomyocytes apoptosis through exosomal miR-21 by targeting PDCD4.

    PubMed

    Xiao, J; Pan, Y; Li, X H; Yang, X Y; Feng, Y L; Tan, H H; Jiang, L; Feng, J; Yu, X Y

    2016-01-01

    Cardiac progenitor cells derived from adult heart have emerged as one of the most promising stem cell types for cardiac protection and repair. Exosomes are known to mediate cell-cell communication by transporting cell-derived proteins and nucleic acids, including various microRNAs (miRNAs). Here we investigated the cardiac progenitor cell (CPC)-derived exosomal miRNAs on protecting myocardium under oxidative stress. Sca1(+)CPCs-derived exosomes were purified from conditional medium, and identified by nanoparticle trafficking analysis (NTA), transmission electron microscopy and western blotting using CD63, CD9 and Alix as markers. Exosomes production was measured by NTA, the result showed that oxidative stress-induced CPCs secrete more exosomes compared with normal condition. Although six apoptosis-related miRNAs could be detected in two different treatment-derived exosomes, only miR-21 was significantly upregulated in oxidative stress-induced exosomes compared with normal exosomes. The same oxidative stress could cause low miR-21 and high cleaved caspase-3 expression in H9C2 cardiac cells. But the cleaved caspase-3 was significantly decreased when miR-21 was overexpressed by transfecting miR-21 mimic. Furthermore, miR-21 mimic or inhibitor transfection and luciferase activity assay confirmed that programmed cell death 4 (PDCD4) was a target gene of miR-21, and miR-21/PDCD4 axis has an important role in anti-apoptotic effect of H9C2 cell. Western blotting and Annexin V/PI results demonstrated that exosomes pre-treated H9C2 exhibited increased miR-21 whereas decreased PDCD4, and had more resistant potential to the apoptosis induced by the oxidative stress, compared with non-treated cells. These findings revealed that CPC-derived exosomal miR-21 had an inhibiting role in the apoptosis pathway through downregulating PDCD4. Restored miR-21/PDCD4 pathway using CPC-derived exosomes could protect myocardial cells against oxidative stress-related apoptosis. Therefore

  6. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy

    NASA Technical Reports Server (NTRS)

    Askari, Arman T.; Unzek, Samuel; Popovic, Zoran B.; Goldman, Corey K.; Forudi, Farhad; Kiedrowski, Matthew; Rovner, Aleksandr; Ellis, Stephen G.; Thomas, James D.; DiCorleto, Paul E.; Topol, Eric J.; Penn, Marc S.

    2003-01-01

    BACKGROUND: Myocardial regeneration via stem-cell mobilisation at the time of myocardial infarction is known to occur, although the mechanism for stem-cell homing to infarcted tissue subsequently and whether this approach can be used for treatment of ischaemic cardiomyopathy are unknown. We investigated these issues in a Lewis rat model (ligation of the left anterior descending artery) of ischaemic cardiomyopathy. METHODS: We studied the effects of stem-cell mobilisation by use of granulocyte colony-stimulating factor (filgrastim) with or without transplantation of syngeneic cells. Shortening fraction and myocardial strain by tissue doppler imaging were quantified by echocardiography. FINDINGS: Stem-cell mobilisation with filgrastim alone did not lead to engraftment of bone-marrow-derived cells. Stromal-cell-derived factor 1 (SDF-1), required for stem-cell homing to bone marrow, was upregulated immediately after myocardial infarction and downregulated within 7 days. 8 weeks after myocardial infarction, transplantation into the peri-infarct zone of syngeneic cardiac fibroblasts stably transfected to express SDF-1 induced homing of CD117-positive stem cells to injured myocardium after filgrastim administration (control vs SDF-1-expressing cardiac fibroblasts mean 7.2 [SD 3.4] vs 33.2 [6.0] cells/mm2, n=4 per group, p<0.02) resulting in greater left-ventricular mass (1.24 [0.29] vs 1.57 [0.27] g) and better cardiac function (shortening fraction 9.2 [4.9] vs 17.2 [4.2]%, n=8 per group, p<0.05). INTERPRETATION: These findings show that SDF-1 is sufficient to induce therapeutic stem-cell homing to injured myocardium and suggest a strategy for directed stem-cell engraftment into injured tissues. Our findings also indicate that therapeutic strategies focused on stem-cell mobilisation for regeneration of myocardial tissue must be initiated within days of myocardial infarction unless signalling for stem-cell homing is re-established.

  7. Stem Cell-Derived Exosomes: A Potential Alternative Therapeutic Agent in Orthopaedics

    PubMed Central

    Burke, John; Kolhe, Ravindra; Hunter, Monte; Isales, Carlos; Hamrick, Mark; Fulzele, Sadanand

    2016-01-01

    Within the field of regenerative medicine, many have sought to use stem cells as a promising way to heal human tissue; however, in the past few years, exosomes (packaged vesicles released from cells) have shown more exciting promise. Specifically, stem cell-derived exosomes have demonstrated great ability to provide therapeutical benefits. Exosomal products can include miRNA, other genetic products, proteins, and various factors. They are released from cells in a paracrine fashion in order to combat local cellular stress. Because of this, there are vast benefits that medicine can obtain from stem cell-derived exosomes. If exosomes could be extracted from stem cells in an efficient manner and packaged with particular regenerative products, then diseases such as rheumatoid arthritis, osteoarthritis, bone fractures, and other maladies could be treated with cell-free regenerative medicine via exosomes. Many advances must be made to get to this point, and the following review highlights the current advances of stem cell-derived exosomes with particular attention to regenerative medicine in orthopaedics. PMID:26904130

  8. Stem Cell-Derived Exosomes: A Potential Alternative Therapeutic Agent in Orthopaedics.

    PubMed

    Burke, John; Kolhe, Ravindra; Hunter, Monte; Isales, Carlos; Hamrick, Mark; Fulzele, Sadanand

    2016-01-01

    Within the field of regenerative medicine, many have sought to use stem cells as a promising way to heal human tissue; however, in the past few years, exosomes (packaged vesicles released from cells) have shown more exciting promise. Specifically, stem cell-derived exosomes have demonstrated great ability to provide therapeutical benefits. Exosomal products can include miRNA, other genetic products, proteins, and various factors. They are released from cells in a paracrine fashion in order to combat local cellular stress. Because of this, there are vast benefits that medicine can obtain from stem cell-derived exosomes. If exosomes could be extracted from stem cells in an efficient manner and packaged with particular regenerative products, then diseases such as rheumatoid arthritis, osteoarthritis, bone fractures, and other maladies could be treated with cell-free regenerative medicine via exosomes. Many advances must be made to get to this point, and the following review highlights the current advances of stem cell-derived exosomes with particular attention to regenerative medicine in orthopaedics. PMID:26904130

  9. Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes.

    PubMed

    Eng, George; Lee, Benjamin W; Protas, Lev; Gagliardi, Mark; Brown, Kristy; Kass, Robert S; Keller, Gordon; Robinson, Richard B; Vunjak-Novakovic, Gordana

    2016-01-01

    The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration. PMID:26785135

  10. Autonomous beating rate adaptation in human stem cell-derived cardiomyocytes

    PubMed Central

    Eng, George; Lee, Benjamin W.; Protas, Lev; Gagliardi, Mark; Brown, Kristy; Kass, Robert S.; Keller, Gordon; Robinson, Richard B.; Vunjak-Novakovic, Gordana

    2016-01-01

    The therapeutic success of human stem cell-derived cardiomyocytes critically depends on their ability to respond to and integrate with the surrounding electromechanical environment. Currently, the immaturity of human cardiomyocytes derived from stem cells limits their utility for regenerative medicine and biological research. We hypothesize that biomimetic electrical signals regulate the intrinsic beating properties of cardiomyocytes. Here we show that electrical conditioning of human stem cell-derived cardiomyocytes in three-dimensional culture promotes cardiomyocyte maturation, alters their automaticity and enhances connexin expression. Cardiomyocytes adapt their autonomous beating rate to the frequency at which they were stimulated, an effect mediated by the emergence of a rapidly depolarizing cell population, and the expression of hERG. This rate-adaptive behaviour is long lasting and transferable to the surrounding cardiomyocytes. Thus, electrical conditioning may be used to promote cardiomyocyte maturation and establish their automaticity, with implications for cell-based reduction of arrhythmia during heart regeneration. PMID:26785135

  11. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

    PubMed Central

    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2014-01-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives. PMID:24232694

  12. Slow conduction in mixed cultured strands of primary ventricular cells and stem cell-derived cardiomyocytes

    PubMed Central

    Kucera, Jan P.; Prudat, Yann; Marcu, Irene C.; Azzarito, Michela; Ullrich, Nina D.

    2015-01-01

    Modern concepts for the treatment of myocardial diseases focus on novel cell therapeutic strategies involving stem cell-derived cardiomyocytes (SCMs). However, functional integration of SCMs requires similar electrophysiological properties as primary cardiomyocytes (PCMs) and the ability to establish intercellular connections with host myocytes in order to contribute to the electrical and mechanical activity of the heart. The aim of this project was to investigate the properties of cardiac conduction in a co-culture approach using SCMs and PCMs in cultured cell strands. Murine embryonic SCMs were pooled with fetal ventricular cells and seeded in predefined proportions on microelectrode arrays to form patterned strands of mixed cells. Conduction velocity (CV) was measured during steady state pacing. SCM excitability was estimated from action potentials measured in single cells using the patch clamp technique. Experiments were complemented with computer simulations of conduction using a detailed model of cellular architecture in mixed cell strands. CV was significantly lower in strands composed purely of SCMs (5.5 ± 1.5 cm/s, n = 11) as compared to PCMs (34.9 ± 2.9 cm/s, n = 21) at similar refractoriness (100% SCMs: 122 ± 25 ms, n = 9; 100% PCMs: 139 ± 67 ms, n = 14). In mixed strands combining both cell types, CV was higher than in pure SCMs strands, but always lower than in 100% PCM strands. Computer simulations demonstrated that both intercellular coupling and electrical excitability limit CV. These data provide evidence that in cultures of murine ventricular cardiomyocytes, SCMs cannot restore CV to control levels resulting in slow conduction, which may lead to reentry circuits and arrhythmias. PMID:26442264

  13. In-vitro stem cell derived red blood cells for transfusion: are we there yet?

    PubMed

    Kim, Hyun Ok

    2014-03-01

    To date, the use of red blood cells (RBCs) produced from stem cells in vitro has not proved practical for routine transfusion. However, the perpetual and widespread shortage of blood products, problems related to transfusion-transmitted infections, and new emerging pathogens elicit an increasing demand for artificial blood. Worldwide efforts to achieve the goal of RBC production through stem cell research have received vast attention; however, problems with large-scale production and cost effectiveness have yet to prove practical usefulness. Some progress has been made, though, as cord blood stem cells and embryonic stem cells have shown an ability to differentiate and proliferate, and induced pluripotent stem cells have been shown to be an unlimited source for RBC production. However, transfusion of stem cell-derived RBCs still presents a number of challenges to overcome. This paper will summarize an up to date account of research and advances in stem cell-derived RBCs, delineate our laboratory protocol in producing RBCs from cord blood, and introduce the technological developments and limitations to current RBC production practices. PMID:24532496

  14. In-Vitro Stem Cell Derived Red Blood Cells for Transfusion: Are We There Yet?

    PubMed Central

    2014-01-01

    To date, the use of red blood cells (RBCs) produced from stem cells in vitro has not proved practical for routine transfusion. However, the perpetual and widespread shortage of blood products, problems related to transfusion-transmitted infections, and new emerging pathogens elicit an increasing demand for artificial blood. Worldwide efforts to achieve the goal of RBC production through stem cell research have received vast attention; however, problems with large-scale production and cost effectiveness have yet to prove practical usefulness. Some progress has been made, though, as cord blood stem cells and embryonic stem cells have shown an ability to differentiate and proliferate, and induced pluripotent stem cells have been shown to be an unlimited source for RBC production. However, transfusion of stem cell-derived RBCs still presents a number of challenges to overcome. This paper will summarize an up to date account of research and advances in stem cell-derived RBCs, delineate our laboratory protocol in producing RBCs from cord blood, and introduce the technological developments and limitations to current RBC production practices. PMID:24532496

  15. Angiogenic activity mediates bone repair from human pluripotent stem cell-derived osteogenic cells

    PubMed Central

    Zou, Li; Chen, Qingshan; Quanbeck, Zachary; Bechtold, Joan E.; Kaufman, Dan S.

    2016-01-01

    Human pluripotent stem cells provide a standardized resource for bone repair. However, criteria to determine which exogenous cells best heal orthopedic injuries remain poorly defined. We evaluated osteogenic progenitor cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs). Phenotypic and genotypic analyses demonstrated that these hESCs/hiPSCs are similar in their osteogenic differentiation efficiency and they generate osteogenic cells comparable to osteogenic cells derived from mesenchymal stromal cells (BM-MSCs). However, expression of angiogenic factors, such as vascular endothelial growth factor and basic fibroblast growth factor in these osteogenic progenitor cells are markedly different, suggesting distinct pro-angiogenic potential of these stem cell derivatives. Studies to repair a femur non-union fracture demonstrate only osteogenic progenitor cells with higher pro-angiogenic potential significantly enhance bone repair in vivo. Together, these studies highlight a key role of pro-angiogenic potential of transplanted osteogenic cells for effective cell-mediated bone repair. PMID:26980556

  16. A Stem Cell-Derived Platform for Studying Single Synaptic Vesicles in Dopaminergic Synapses

    PubMed Central

    Gu, Haigang; Lazarenko, Roman M.; Koktysh, Dmitry; Iacovitti, Lorraine

    2015-01-01

    The exocytotic release of dopamine is one of the most characteristic but also one of the least appreciated processes in dopaminergic neurotransmission. Fluorescence imaging has yielded rich information about the properties of synaptic vesicles and the release of neurotransmitters in excitatory and inhibitory neurons. In contrast, imaging-based studies for in-depth understanding of synaptic vesicle behavior in dopamine neurons are lagging largely because of a lack of suitable preparations. Midbrain culture has been one of the most valuable preparations for the subcellular investigation of dopaminergic transmission; however, the paucity and fragility of cultured dopaminergic neurons limits their use for live cell imaging. Recent developments in stem cell technology have led to the successful production of dopamine neurons from embryonic or induced pluripotent stem cells. Although the dopaminergic identity of these stem cell-derived neurons has been characterized in different ways, vesicle-mediated dopamine release from their axonal terminals has been barely assessed. We report a more efficient procedure to reliably generate dopamine neurons from embryonic stem cells, and it yields more dopamine neurons with more dopaminergic axon projections than midbrain culture does. Using a collection of functional measurements, we show that stem cell-derived dopamine neurons are indistinguishable from those in midbrain culture. Taking advantage of this new preparation, we simultaneously tracked the turnover of hundreds of synaptic vesicles individually using pH-sensitive quantum dots. By doing so, we revealed distinct fusion kinetics of the dopamine-secreting vesicles, which is consistent within both preparations. Significance For the use of stem cell-derived neurons in clinical applications, improved differentiation efficiency and more careful characterization of resultant cells are needed. A procedure has been refined for differentiation of mouse embryonic stem cells into

  17. Controversies in Cardiovascular Research: Induced pluripotent stem cell-derived cardiomyocytes – boutique science or valuable arrhythmia model?

    PubMed Central

    Knollmann, Björn C

    2013-01-01

    As part of the series on Controversies in Cardiovascular Research, the article reviews the strengths and limitations of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) as models of cardiac arrhythmias. Specifically, the article attempts to answer the following questions: Which clinical arrhythmias can be modeled by iPSC-CM? How well can iPSC-CM model adult ventricular myocytes? What are the strengths and limitations of published iPSC-CM arrhythmia models? What new mechanistic insight has been gained? What is the evidence that would support using iPSC-CM to personalize anti-arrhythmic drug therapy? The review also discusses the pros and cons of using the iPSC-CM technology for modeling specific genetic arrhythmia disorders such as long QT syndrome, Brugada Syndrome or Catecholaminergic Polymorphic Ventricular Tachycardia. PMID:23569106

  18. Concise review: the relevance of human stem cell-derived organoid models for epithelial translational medicine.

    PubMed

    Hynds, Robert E; Giangreco, Adam

    2013-03-01

    Epithelial organ remodeling is a major contributing factor to worldwide death and disease, costing healthcare systems billions of dollars every year. Despite this, most fundamental epithelial organ research fails to produce new therapies and mortality rates for epithelial organ diseases remain unacceptably high. In large part, this failure in translating basic epithelial research into clinical therapy is due to a lack of relevance in existing preclinical models. To correct this, new models are required that improve preclinical target identification, pharmacological lead validation, and compound optimization. In this review, we discuss the relevance of human stem cell-derived, three-dimensional organoid models for addressing each of these challenges. We highlight the advantages of stem cell-derived organoid models over existing culture systems, discuss recent advances in epithelial tissue-specific organoids, and present a paradigm for using organoid models in human translational medicine. PMID:23203919

  19. Generation of Highly Purified Human Cardiomyocytes from Peripheral Blood Mononuclear Cell-Derived Induced Pluripotent Stem Cells

    PubMed Central

    Stark, Klaus; Jentsch, Nico; Klingenstein, Melanie; Drzymalski, Marzena; Wagner, Stefan; Maier, Lars S.; Hehr, Ute; Baessler, Andrea; Fischer, Marcus; Hengstenberg, Christian

    2015-01-01

    Induced pluripotent stem (iPS) cells have an enormous potential for physiological studies. A novel protocol was developed combining the derivation of iPS from peripheral blood with an optimized directed differentiation to cardiomyocytes and a subsequent metabolic selection. The human iPS cells were retrovirally dedifferentiated from activated T cells. The subsequent optimized directed differentiation protocol yielded 30-45% cardiomyocytes at day 16 of differentiation. The derived cardiomyocytes expressed appropriate structural markers like cardiac troponin T, α-actinin and myosin light chain 2 (MLC2V). In a subsequent metabolic selection with lactate, the cardiomyocytes content could be increased to more than 90%. Loss of cardiomyocytes during metabolic selection were less than 50%, whereas alternative surface antibody-based selection procedures resulted in loss of up to 80% of cardiomyocytes. Electrophysiological characterization confirmed the typical cardiac features and the presence of ventricular, atrial and nodal-like action potentials within the derived cardiomyocyte population. Our combined and optimized protocol is highly robust and applicable for scalable cardiac differentiation. It provides a simple and cost-efficient method without expensive equipment for generating large numbers of highly purified, functional cardiomyocytes. It will further enhance the applicability of iPS cell-derived cardiomyocytes for disease modeling, drug discovery, and regenerative medicine. PMID:25970162

  20. Functional Properties of Human Stem Cell-Derived Neurons in Health and Disease

    PubMed Central

    Weick, Jason P.

    2016-01-01

    Stem cell-derived neurons from various source materials present unique model systems to examine the fundamental properties of central nervous system (CNS) development as well as the molecular underpinnings of disease phenotypes. In order to more accurately assess potential therapies for neurological disorders, multiple strategies have been employed in recent years to produce neuronal populations that accurately represent in vivo regional and transmitter phenotypes. These include new technologies such as direct conversion of somatic cell types into neurons and glia which may accelerate maturation and retain genetic hallmarks of aging. In addition, novel forms of genetic manipulations have brought human stem cells nearly on par with those of rodent with respect to gene targeting. For neurons of the CNS, the ultimate phenotypic characterization lies with their ability to recapitulate functional properties such as passive and active membrane characteristics, synaptic activity, and plasticity. These features critically depend on the coordinated expression and localization of hundreds of ion channels and receptors, as well as scaffolding and signaling molecules. In this review I will highlight the current state of knowledge regarding functional properties of human stem cell-derived neurons, with a primary focus on pluripotent stem cells. While significant advances have been made, critical hurdles must be overcome in order for this technology to support progression toward clinical applications. PMID:27274733

  1. Drug-loaded nanoparticles induce gene expression in human pluripotent stem cell derivatives

    NASA Astrophysics Data System (ADS)

    Gajbhiye, Virendra; Escalante, Leah; Chen, Guojun; Laperle, Alex; Zheng, Qifeng; Steyer, Benjamin; Gong, Shaoqin; Saha, Krishanu

    2013-12-01

    Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained higher fibroblast proliferation levels and MMP activity. The results demonstrate that the PEG-H40-DXC nanoparticle system provides an effective tool to controlling gene expression in human stem cell derivatives.Tissue engineering and advanced manufacturing of human stem cells requires a suite of tools to control gene expression spatiotemporally in culture. Inducible gene expression systems offer cell-extrinsic control, typically through addition of small molecules, but small molecule inducers typically contain few functional groups for further chemical modification. Doxycycline (DXC), a potent small molecule inducer of tetracycline (Tet) transgene systems, was conjugated to a hyperbranched dendritic polymer (Boltorn H40) and subsequently reacted with polyethylene glycol (PEG). The resulting PEG-H40-DXC nanoparticle exhibited pH-sensitive drug release behavior and successfully controlled gene expression in stem-cell-derived fibroblasts with a Tet-On system. While free DXC inhibited fibroblast proliferation and matrix metalloproteinase (MMP) activity, PEG-H40-DXC nanoparticles maintained

  2. Stem cell-derived nodal-like cardiomyocytes as a novel pharmacologic tool: insights from sinoatrial node development and function.

    PubMed

    Barbuti, Andrea; Robinson, Richard B

    2015-01-01

    Since the first reports on the isolation and differentiation of stem cells, and in particular since the early success in driving these cells down a cardiac lineage, there has been interest in the potential of such preparations in cardiac regenerative therapy. Much of the focus of such research has been on improving mechanical function after myocardial infarction; however, electrophysiologic studies of these preparations have revealed a heterogeneous mix of action potential characteristics, including some described as "pacemaker" or "nodal-like," which in turn led to interest in the therapeutic potential of these preparations in the treatment of rhythm disorders; several proof-of-concept studies have used these cells to create a biologic alternative to electronic pacemakers. Further, there are additional potential applications of a preparation of pacemaker cells derived from stem cells, for example, in high-throughput screens of new chronotropic agents. All such applications require reasonably efficient methods for selecting or enriching the "nodal-like" cells, however, which in turn depends on first defining what constitutes a nodal-like cell since not all pacemaking cells are necessarily of nodal lineage. This review discusses the current state of the field in terms of characterizing sinoatrial-like cardiomyocytes derived from embryonic and induced pluripotent stem cells, markers that might be appropriate based on the current knowledge of the gene program leading to sinoatrial node development, what functional characteristics might be expected and desired based on studies of the sinoatrial node, and recent efforts at enrichment and selection of nodal-like cells. PMID:25733770

  3. Mesenchymal Stem Cells with Increased Stromal Cell-Derived Factor 1 Expression Enhanced Fracture Healing

    PubMed Central

    Ho, Chih-Yuan; Hua, Jia; Coathup, Melanie; Kalia, Priya; Blunn, Gordon

    2015-01-01

    Treatment of critical size bone defects pose a challenge in orthopedics. Stem cell therapy together with cytokines has the potential to improve bone repair as they cause the migration and homing of stem cells to the defect site. However, the engraftment, participation, and recruitment of other cells within the regenerating tissue are important. To enhance stem cell involvement, this study investigated overexpression of stem cells with stromal cell-derived factor 1 (SDF-1) using an adenovirus. We hypothesized that these engineered cells would effectively increase the migration of native cells to the site of fracture, enhancing bone repair. Before implantation, we showed that SDF-1 secreted by transfected cells increased the migration of nontransfected cells. In a rat defect bone model, bone marrow mesenchymal stem cells overexpressing SDF-1 showed significantly (p=0.003) more new bone formation within the gap and less bone mineral loss at the area adjacent to the defect site during the early bone healing stage. In conclusion, SDF-1 was shown to play an important role in accelerating fracture repair and contributing to bone repair in rat models, by recruiting more host stem cells to the defect site and encouraging osteogenic differentiation and production of bone. PMID:25251779

  4. Hepatic Cells Derived from Induced Pluripotent Stem Cells of Pigtail Macaques Support Hepatitis C Virus infection

    PubMed Central

    Sourisseau, Marion; Goldman, Orit; He, Wenqian; Gori, Jennifer L.; Kiem, Hans-Peter; Gouon-Evans, Valerie; Evans, Matthew J.

    2013-01-01

    The narrow species tropism of hepatitis C virus (HCV) limits animal studies. We found that pigtail macaque (Macaca nemestrina) hepatic cells derived from induced pluripotent stem cells support the entire HCV life cycle, although infection efficiency was limited by defects in the HCV cell entry process. This block was overcome by either increasing occludin expression, complementing the cells with human CD81, or infecting them with a strain of HCV with less-restricted requirements for CD81. Using this system, we can modify viral and host cell genetics to make pigtail macaques a suitable, clinically relevant model for the study of HCV infection. PMID:23891978

  5. Advancements in Induced Pluripotent Stem Cell Technology for Cardiac Regenerative Medicine

    PubMed Central

    Suh, Carol Y.; Wang, Zelun; Bártulos, Oscar; Qyang, Yibing

    2014-01-01

    Cardiovascular diseases remain the leading causes of morbidity and mortality in the developed world. Cellular based cardiac regenerative therapy serves as a potential approach to treating cardiovascular diseases. Although various cellular types have been tested, induced pluripotent stem cells are regarded as a promising cell source for therapy. In this review, we will highlight some of the advances in generating induced pluripotent stem cells and differentiation to cardiac cells. We will also discuss the progress in modeling cardiovascular diseases using induced pluripotent stem cell derived cardiac cells. As we continue to make progress in induced pluripotent stem cell and cardiac differentiation technology, we will become closer to application of cardiac regenerative medicine. PMID:24651517

  6. Comparison of Magnetic Resonance Imaging and Serum Biomarkers for Detection of Human Pluripotent Stem Cell-Derived Teratomas

    PubMed Central

    Riegler, Johannes; Ebert, Antje; Qin, Xulei; Shen, Qi; Wang, Mouer; Ameen, Mohamed; Kodo, Kazuki; Ong, Sang-Ging; Lee, Won Hee; Lee, Grace; Neofytou, Evgenios; Gold, Joseph D.; Connolly, Andrew J.; Wu, Joseph C.

    2016-01-01

    Summary The use of cells derived from pluripotent stem cells (PSCs) for regenerative therapies confers a considerable risk for neoplastic growth and teratoma formation. Preclinical and clinical assessment of such therapies will require suitable monitoring strategies to understand and mitigate these risks. Here we generated human-induced pluripotent stem cells (iPSCs), selected clones that continued to express reprogramming factors after differentiation into cardiomyocytes, and transplanted these cardiomyocytes into immunocompromised rat hearts post-myocardial infarction. We compared magnetic resonance imaging (MRI), cardiac ultrasound, and serum biomarkers for their ability to delineate teratoma formation and growth. MRI enabled the detection of teratomas with a volume >8 mm3. A combination of three plasma biomarkers (CEA, AFP, and HCG) was able to detect teratomas with a volume >17 mm3 and with a sensitivity of more than 87%. Based on our findings, a combination of serum biomarkers with MRI screening may offer the highest sensitivity for teratoma detection and tracking. PMID:26777057

  7. Structural and functional screening in human induced-pluripotent stem cell-derived cardiomyocytes accurately identifies cardiotoxicity of multiple drug types

    SciTech Connect

    Doherty, Kimberly R. Talbert, Dominique R.; Trusk, Patricia B.; Moran, Diarmuid M.; Shell, Scott A.; Bacus, Sarah

    2015-05-15

    Safety pharmacology studies that evaluate new drug entities for potential cardiac liability remain a critical component of drug development. Current studies have shown that in vitro tests utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) may be beneficial for preclinical risk evaluation. We recently demonstrated that an in vitro multi-parameter test panel assessing overall cardiac health and function could accurately reflect the associated clinical cardiotoxicity of 4 FDA-approved targeted oncology agents using hiPS-CM. The present studies expand upon this initial observation to assess whether this in vitro screen could detect cardiotoxicity across multiple drug classes with known clinical cardiac risks. Thus, 24 drugs were examined for their effect on both structural (viability, reactive oxygen species generation, lipid formation, troponin secretion) and functional (beating activity) endpoints in hiPS-CM. Using this screen, the cardiac-safe drugs showed no effects on any of the tests in our panel. However, 16 of 18 compounds with known clinical cardiac risk showed drug-induced changes in hiPS-CM by at least one method. Moreover, when taking into account the Cmax values, these 16 compounds could be further classified depending on whether the effects were structural, functional, or both. Overall, the most sensitive test assessed cardiac beating using the xCELLigence platform (88.9%) while the structural endpoints provided additional insight into the mechanism of cardiotoxicity for several drugs. These studies show that a multi-parameter approach examining both cardiac cell health and function in hiPS-CM provides a comprehensive and robust assessment that can aid in the determination of potential cardiac liability. - Highlights: • 24 drugs were tested for cardiac liability using an in vitro multi-parameter screen. • Changes in beating activity were the most sensitive in predicting cardiac risk. • Structural effects add in

  8. Stem cell-derived motor neurons from spinal and bulbar muscular atrophy patients.

    PubMed

    Grunseich, Christopher; Zukosky, Kristen; Kats, Ilona R; Ghosh, Laboni; Harmison, George G; Bott, Laura C; Rinaldi, Carlo; Chen, Ke-lian; Chen, Guibin; Boehm, Manfred; Fischbeck, Kenneth H

    2014-10-01

    Spinal and bulbar muscular atrophy (SBMA, Kennedy's disease) is a motor neuron disease caused by polyglutamine repeat expansion in the androgen receptor. Although degeneration occurs in the spinal cord and muscle, the exact mechanism is not clear. Induced pluripotent stem cells from spinal and bulbar muscular atrophy patients provide a useful model for understanding the disease mechanism and designing effective therapy. Stem cells were generated from six patients and compared to control lines from three healthy individuals. Motor neurons from four patients were differentiated from stem cells and characterized to understand disease-relevant phenotypes. Stem cells created from patient fibroblasts express less androgen receptor than control cells, but show androgen-dependent stabilization and nuclear translocation. The expanded repeat in several stem cell clones was unstable, with either expansion or contraction. Patient stem cell clones produced a similar number of motor neurons compared to controls, with or without androgen treatment. The stem cell-derived motor neurons had immunoreactivity for HB9, Isl1, ChAT, and SMI-32, and those with the largest repeat expansions were found to have increased acetylated α-tubulin and reduced HDAC6. Reduced HDAC6 was also found in motor neuron cultures from two other patients with shorter repeats. Evaluation of stably transfected mouse cells and SBMA spinal cord showed similar changes in acetylated α-tubulin and HDAC6. Perinuclear lysosomal enrichment, an HDAC6 dependent process, was disrupted in motor neurons from two patients with the longest repeats. SBMA stem cells present new insights into the disease, and the observations of reduced androgen receptor levels, repeat instability, and reduced HDAC6 provide avenues for further investigation of the disease mechanism and development of effective therapy. PMID:24925468

  9. Radioresistance of cancer stem-like cell derived from canine tumours.

    PubMed

    Tanabe, A; Deguchi, T; Sato, T; Nemoto, Y; Maruo, T; Madarame, H; Shida, T; Naya, Y; Ogihara, K; Sahara, H

    2016-09-01

    Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are a small subpopulation of cancer cells that are responsible for the initiation, recurrence and metastasis of cancer. We previously demonstrated that, using the Hoechst 33342 dye-based side population technique, CSCs/CICs in canine lung adenocarcinoma cell line exist. In this study, as CSCs/CICs are known to form spheres in anchorage-independent environment in vitro, we evaluated the stemness of spheroid cells derived from canine lung adenocarcinoma and osteosarcoma cells by expression of stemness markers, and investigated radioresistance. Spheroid cells showed greater expression of stemness markers Oct-4 and CD133 gene than those of adherent-cultured cells. In nude mouse xenograft models, spheroid cells showed higher tumourigenic ability than adherent-cultured cells. In addition, spheroid cells showed significantly resistant against radioactivity as compared with adherent-cultured cells. These results suggest that spheroid cells could possess stemness and provide a CSCs/CICs research tool to investigate CSCs/CICs of canine tumour cells. PMID:25070729

  10. Stimulation of human embryonic stem cell-derived cardiomyocytes on thin-film microelectrodes.

    PubMed

    Viitanen, Jouko; Heimala, Päivi; Hokkanen, Ari; Iljin, Kristiina; Kerkelä, Erja; Kolari, Kai; Kattelus, Hannu

    2011-05-01

    We describe successful long-term stimulation of human embryonic stem cell-derived cardiomyocyte clusters on thin-film microelectrode structures in vitro. Interdigitated electrode structures were constructed using plain titanium on glass as the electrode material. Titanium rapidly oxidizes in atmospheric conditions to produce an insulating TiO(χ) layer with high relative permittivity. Capacitive coupling to the incubation medium and to the cells adherent to the electrodes was still efficient, and the dielectric layer prevented electrolysis, allowing a wider window of possible stimulation amplitudes to be used, relative to conducting surfaces. A common hypothesis suggests that to achieve proper differentiation of electroactive cells from the stem cells electrical stimuli are also needed. Spontaneously beating cardiomyocyte clusters were seeded on the glass-electrode surfaces, and we successfully altered and resynchronized a clearly different beat interval. The new pace was reliably maintained for extended periods of several tens of minutes. PMID:21416608

  11. Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

    PubMed Central

    Bayzigitov, Daniel R.; Medvedev, Sergey P.; Dementyeva, Elena V.; Bayramova, Sevda A.; Pokushalov, Evgeny A.; Karaskov, Alexander M.; Zakian, Suren M.

    2016-01-01

    Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes. PMID:27110425

  12. Effects of tacrolimus on morphology, proliferation and differentiation of mesenchymal stem cells derived from gingiva tissue.

    PubMed

    Ha, Dong-Ho; Yong, Chul Soon; Kim, Jong Oh; Jeong, Jee-Heon; Park, Jun-Beom

    2016-07-01

    Tacrolimus is a 23-membered macrolide lactone with potent immunosuppressive activity that is effective in the prophylaxis of organ rejection following kidney, heart and liver transplantation. Tacrolimus also exerts a variety of actions on bone metabolism. The aim of the present study was to evaluate the effects of different concentrations of tacrolimus on the morphology and viability of human stem cells derived from the gingiva. Gingival‑derived stem cells were grown in the presence of tacrolimus at final concentrations ranging from 0.001 to 100 µg/ml. The morphology of the cells was viewed under an inverted microscope and the cell viability was analyzed using Cell Counting kit‑8 (CCK‑8) on days 1, 3, 5 and 7. Alizarin Red S staining was used to assess mineralization of treated cells. The control group showed spindle‑shaped, fibroblast‑like morphology and the shapes of the cells in 0.001, 0.01, 0.1, 1 and 10 µg/ml tacrolimus were similar to those of the control group. All groups except the 100 µg/ml group showed increased cell proliferation over time. Cultures grown in the presence of tacrolimus at 0.001, 0.01, 0.1, 1 and 10 µg/ml were not identified to be significantly different compared with the control at days 1, 3 and 5 using the CCK‑8 assays. Increased mineralized deposits were noted with increased incubation time. Treatment with tacrolimus from 0.001 to 1 µg/ml led to an increase in mineralization compared with the control group. Within the limits of this study, tacrolimus at the tested concentrations (ranging from 0.001 to 10 µg/ml) did not result in differences in the viability of stem cells derived from gingiva; however it did enhance osteogenic differentiation of the stem cells. PMID:27177273

  13. Effects of tacrolimus on morphology, proliferation and differentiation of mesenchymal stem cells derived from gingiva tissue

    PubMed Central

    HA, DONG-HO; YONG, CHUL SOON; KIM, JONG OH; JEONG, JEE-HEON; PARK, JUN-BEOM

    2016-01-01

    Tacrolimus is a 23-membered macrolide lactone with potent immunosuppressive activity that is effective in the prophylaxis of organ rejection following kidney, heart and liver transplantation. Tacrolimus also exerts a variety of actions on bone metabolism. The aim of the present study was to evaluate the effects of different concentrations of tacrolimus on the morphology and viability of human stem cells derived from the gingiva. Gingival-derived stem cells were grown in the presence of tacrolimus at final concentrations ranging from 0.001 to 100 µg/ml. The morphology of the cells was viewed under an inverted microscope and the cell viability was analyzed using Cell Counting kit-8 (CCK-8) on days 1, 3, 5 and 7. Alizarin Red S staining was used to assess mineralization of treated cells. The control group showed spindle-shaped, fibroblast-like morphology and the shapes of the cells in 0.001, 0.01, 0.1, 1 and 10 µg/ml tacrolimus were similar to those of the control group. All groups except the 100 µg/ml group showed increased cell proliferation over time. Cultures grown in the presence of tacrolimus at 0.001, 0.01, 0.1, 1 and 10 µg/ml were not identified to be significantly different compared with the control at days 1, 3 and 5 using the CCK-8 assays. Increased mineralized deposits were noted with increased incubation time. Treatment with tacrolimus from 0.001 to 1 µg/ml led to an increase in mineralization compared with the control group. Within the limits of this study, tacrolimus at the tested concentrations (ranging from 0.001 to 10 µg/ml) did not result in differences in the viability of stem cells derived from gingiva; however it did enhance osteogenic differentiation of the stem cells. PMID:27177273

  14. Suppression of Th1-Mediated Autoimmunity by Embryonic Stem Cell-Derived Dendritic Cells

    PubMed Central

    Ikeda, Tokunori; Hirata, Shinya; Takamatsu, Koutaro; Haruta, Miwa; Tsukamoto, Hirotake; Ito, Takaaki; Uchino, Makoto; Ando, Yukio; Nagafuchi, Seiho; Nishimura, Yasuharu; Senju, Satoru

    2014-01-01

    We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs) using two models of autoimmune disease, namely non-obese diabetic (NOD) mice and experimental autoimmune encephalomyelitis (EAE). Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases. PMID:25522369

  15. Suppression of Th1-mediated autoimmunity by embryonic stem cell-derived dendritic cells.

    PubMed

    Ikeda, Tokunori; Hirata, Shinya; Takamatsu, Koutaro; Haruta, Miwa; Tsukamoto, Hirotake; Ito, Takaaki; Uchino, Makoto; Ando, Yukio; Nagafuchi, Seiho; Nishimura, Yasuharu; Senju, Satoru

    2014-01-01

    We herein demonstrate the immune-regulatory effect of embryonic stem cell-derived dendritic cells (ES-DCs) using two models of autoimmune disease, namely non-obese diabetic (NOD) mice and experimental autoimmune encephalomyelitis (EAE). Treatment of pre-diabetic NOD mice with ES-DCs exerted almost complete suppression of diabetes development during the observation period for more than 40 weeks. The prevention of diabetes by ES-DCs was accompanied with significant reduction of insulitis and decreased number of Th1 and Th17 cells in the spleen. Development of EAE was also inhibited by the treatment with ES-DCs, and the therapeutic effect was obtained even if ES-DCs were administrated after the onset of clinical symptoms. Treatment of EAE-induced mice with ES-DCs reduced the infiltration of inflammatory cells into the spinal cord and suppressed the T cell response to the myelin antigen. Importantly, the ES-DC treatment did not affect T cell response to an exogenous antigen. As the mechanisms underlying the reduction of the number of infiltrating Th1 cells, we observed the inhibition of differentiation and proliferation of Th1 cells by ES-DCs. Furthermore, the expression of VLA-4α on Th1 cells was significantly inhibited by ES-DCs. Considering the recent advances in human induced pluripotent stem cell-related technologies, these results suggest a clinical application for pluripotent stem cell-derived dendritic cells as a therapy for T cell-mediated autoimmune diseases. PMID:25522369

  16. Human Induced Pluripotent Stem Cell-Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation.

    PubMed

    Sharmin, Sazia; Taguchi, Atsuhiro; Kaku, Yusuke; Yoshimura, Yasuhiro; Ohmori, Tomoko; Sakuma, Tetsushi; Mukoyama, Masashi; Yamamoto, Takashi; Kurihara, Hidetake; Nishinakamura, Ryuichi

    2016-06-01

    Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator-like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in vitro These induced human podocytes exhibited apicobasal polarity, with nephrin proteins accumulated close to the basal domain, and possessed primary processes that were connected with slit diaphragm-like structures. Microarray analysis of sorted iPS cell-derived podocytes identified well conserved marker gene expression previously shown in mouse and human podocytes in vivo Furthermore, we developed a novel transplantation method using spacers that release the tension of host kidney capsules, thereby allowing the effective formation of glomeruli from human iPS cell-derived nephron progenitors. The human glomeruli were vascularized with the host mouse endothelial cells, and iPS cell-derived podocytes with numerous cell processes accumulated around the fenestrated endothelial cells. Therefore, the podocytes generated from iPS cells retain the podocyte-specific molecular and structural features, which will be useful for dissecting human glomerular development and diseases. PMID:26586691

  17. Generation of stem cell-derived β-cells from patients with type 1 diabetes

    PubMed Central

    Millman, Jeffrey R.; Xie, Chunhui; Van Dervort, Alana; Gürtler, Mads; Pagliuca, Felicia W.; Melton, Douglas A.

    2016-01-01

    We recently reported the scalable in vitro production of functional stem cell-derived β-cells (SC-β cells). Here we extend this approach to generate the first SC-β cells from type 1 diabetic patients (T1D). β-cells are destroyed during T1D disease progression, making it difficult to extensively study them in the past. These T1D SC-β cells express β-cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of β-cell stress. Using these assays, we find no major differences in T1D SC-β cells compared with SC-β cells derived from non-diabetic patients. These results show that T1D SC-β cells could potentially be used for the treatment of diabetes, drug screening and the study of β-cell biology. PMID:27163171

  18. Generation of stem cell-derived β-cells from patients with type 1 diabetes.

    PubMed

    Millman, Jeffrey R; Xie, Chunhui; Van Dervort, Alana; Gürtler, Mads; Pagliuca, Felicia W; Melton, Douglas A

    2016-01-01

    We recently reported the scalable in vitro production of functional stem cell-derived β-cells (SC-β cells). Here we extend this approach to generate the first SC-β cells from type 1 diabetic patients (T1D). β-cells are destroyed during T1D disease progression, making it difficult to extensively study them in the past. These T1D SC-β cells express β-cell markers, respond to glucose both in vitro and in vivo, prevent alloxan-induced diabetes in mice and respond to anti-diabetic drugs. Furthermore, we use an in vitro disease model to demonstrate the cells respond to different forms of β-cell stress. Using these assays, we find no major differences in T1D SC-β cells compared with SC-β cells derived from non-diabetic patients. These results show that T1D SC-β cells could potentially be used for the treatment of diabetes, drug screening and the study of β-cell biology. PMID:27163171

  19. Osteocyte specific responses to soluble and mechanical stimuli in a stem cell derived culture model

    PubMed Central

    Thompson, William R.; Uzer, Gunes; Brobst, Kaitlyn E.; Xie, Zhihui; Sen, Buer; Yen, Sherwin S.; Styner, Maya; Rubin, Janet

    2015-01-01

    Studying osteocyte behavior in culture has proven difficult because these embedded cells require spatially coordinated interactions with the matrix and surrounding cells to achieve the osteocyte phenotype. Using an easily attainable source of bone marrow mesenchymal stem cells, we generated cells with the osteocyte phenotype within two weeks. These “stem cell derived-osteocytes” (SCD-O) displayed stellate morphology and lacunocanalicular ultrastructure. Osteocytic genes Sost, Dmp1, E11, and Fgf23 were maximally expressed at 15 days and responded to PTH and 1,25(OH)2D3. Production of sclerostin mRNA and protein, within 15 days of culture makes the SCD-O model ideal for elucidating regulatory mechanisms. We found sclerostin to be regulated by mechanical factors, where low intensity vibration significantly reduced Sost expression. Additionally, this model recapitulates sclerostin production in response to osteoactive hormones, as PTH or LIV repressed secretion of sclerostin, significantly impacting Wnt-mediated Axin2 expression, via β-catenin signaling. In summary, SCD-O cells produce abundant matrix, rapidly attain the osteocyte phenotype, and secrete functional factors including sclerostin under non-immortalized conditions. This culture model enables ex vivo observations of osteocyte behavior while preserving an organ-like environment. Furthermore, as marrow-derived mesenchymal stem cells can be obtained from transgenic animals; our model enables study of genetic control of osteocyte behaviors. PMID:26056071

  20. Human pluripotent stem cell-derived neural constructs for predicting neural toxicity.

    PubMed

    Schwartz, Michael P; Hou, Zhonggang; Propson, Nicholas E; Zhang, Jue; Engstrom, Collin J; Santos Costa, Vitor; Jiang, Peng; Nguyen, Bao Kim; Bolin, Jennifer M; Daly, William; Wang, Yu; Stewart, Ron; Page, C David; Murphy, William L; Thomson, James A

    2015-10-01

    Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial. PMID:26392547

  1. Potential Therapies by Stem Cell-Derived Exosomes in CNS Diseases: Focusing on the Neurogenic Niche

    PubMed Central

    Luarte, Alejandro; Bátiz, Luis Federico; Wyneken, Ursula; Lafourcade, Carlos

    2016-01-01

    Neurodegenerative disorders are one of the leading causes of death and disability and one of the biggest burdens on health care systems. Novel approaches using various types of stem cells have been proposed to treat common neurodegenerative disorders such as Alzheimer's Disease, Parkinson's Disease, or stroke. Moreover, as the secretome of these cells appears to be of greater benefit compared to the cells themselves, the extracellular components responsible for its therapeutic benefit have been explored. Stem cells, as well as most cells, release extracellular vesicles such as exosomes, which are nanovesicles able to target specific cell types and thus to modify their function by delivering proteins, lipids, and nucleic acids. Exosomes have recently been tested in vivo and in vitro as therapeutic conveyors for the treatment of diseases. As such, they could be engineered to target specific populations of cells within the CNS. Considering the fact that many degenerative brain diseases have an impact on adult neurogenesis, we discuss how the modulation of the adult neurogenic niches may be a therapeutic target of stem cell-derived exosomes. These novel approaches should be examined in cellular and animal models to provide better, more effective, and specific therapeutic tools in the future. PMID:27195011

  2. Accurate Prediction of Drug-Induced Liver Injury Using Stem Cell-Derived Populations

    PubMed Central

    Szkolnicka, Dagmara; Farnworth, Sarah L.; Lucendo-Villarin, Baltasar; Storck, Christopher; Zhou, Wenli; Iredale, John P.; Flint, Oliver

    2014-01-01

    Despite major progress in the knowledge and management of human liver injury, there are millions of people suffering from chronic liver disease. Currently, the only cure for end-stage liver disease is orthotopic liver transplantation; however, this approach is severely limited by organ donation. Alternative approaches to restoring liver function have therefore been pursued, including the use of somatic and stem cell populations. Although such approaches are essential in developing scalable treatments, there is also an imperative to develop predictive human systems that more effectively study and/or prevent the onset of liver disease and decompensated organ function. We used a renewable human stem cell resource, from defined genetic backgrounds, and drove them through developmental intermediates to yield highly active, drug-inducible, and predictive human hepatocyte populations. Most importantly, stem cell-derived hepatocytes displayed equivalence to primary adult hepatocytes, following incubation with known hepatotoxins. In summary, we have developed a serum-free, scalable, and shippable cell-based model that faithfully predicts the potential for human liver injury. Such a resource has direct application in human modeling and, in the future, could play an important role in developing renewable cell-based therapies. PMID:24375539

  3. Muse Cells, a New Type of Pluripotent Stem Cell Derived from Human Fibroblasts.

    PubMed

    Liu, Qi; Zhang, Ru-zhi; Li, Di; Cheng, Sai; Yang, Yu-hua; Tian, Ting; Pan, Xiao-ru

    2016-04-01

    A new type of mesenchymal stem cells (MSCs) that expresses stage-specific embryonic antigen 3 (SSEA-3) and the mesenchymal cell marker CD105 are known as multilineage-differentiating stress-enduring (Muse) cells. Studies have shown that stem cells in suspension cultures are more likely to generate embryoid body-like stem cell spheres and maintain an undifferentiated phenotype and pluripotency. We separated Muse cells derived from human dermal fibroblasts by long-term trypsin incubation (LTT) through suspension cultures in methylcellulose. The Muse cells obtained expressed several pluripotency markers, including Nanog, Oct4, Sox2, and SSEA-3, and could differentiate in vitro into cells of the three germ layers, such as hepatocytes (endodermal), neural cells (ectodermal) and adipocytes, and osteocytes (mesodermal cells). These cells showed a low level of DNA methylation and a high nucleo-cytoplasmic ratio. Our study provides an innovative and exciting platform for exploring the potential cell-based therapy of various human diseases using Muse cells as well as their great possibility for regenerative medicine. PMID:27055628

  4. Human pluripotent stem cell-derived neural constructs for predicting neural toxicity

    PubMed Central

    Schwartz, Michael P.; Hou, Zhonggang; Propson, Nicholas E.; Zhang, Jue; Engstrom, Collin J.; Costa, Vitor Santos; Jiang, Peng; Nguyen, Bao Kim; Bolin, Jennifer M.; Daly, William; Wang, Yu; Stewart, Ron; Page, C. David; Murphy, William L.; Thomson, James A.

    2015-01-01

    Human pluripotent stem cell-based in vitro models that reflect human physiology have the potential to reduce the number of drug failures in clinical trials and offer a cost-effective approach for assessing chemical safety. Here, human embryonic stem (ES) cell-derived neural progenitor cells, endothelial cells, mesenchymal stem cells, and microglia/macrophage precursors were combined on chemically defined polyethylene glycol hydrogels and cultured in serum-free medium to model cellular interactions within the developing brain. The precursors self-assembled into 3D neural constructs with diverse neuronal and glial populations, interconnected vascular networks, and ramified microglia. Replicate constructs were reproducible by RNA sequencing (RNA-Seq) and expressed neurogenesis, vasculature development, and microglia genes. Linear support vector machines were used to construct a predictive model from RNA-Seq data for 240 neural constructs treated with 34 toxic and 26 nontoxic chemicals. The predictive model was evaluated using two standard hold-out testing methods: a nearly unbiased leave-one-out cross-validation for the 60 training compounds and an unbiased blinded trial using a single hold-out set of 10 additional chemicals. The linear support vector produced an estimate for future data of 0.91 in the cross-validation experiment and correctly classified 9 of 10 chemicals in the blinded trial. PMID:26392547

  5. Functional differentiation of stem cell-derived neurons from different murine backgrounds

    PubMed Central

    Barth, Lydia; Sütterlin, Rosmarie; Nenniger, Markus; Vogt, Kaspar E.

    2014-01-01

    Murine stem cell-derived neurons have been used to study a wide variety of neuropsychiatric diseases with a hereditary component, ranging from autism to Alzheimer’s. While a significant amount of data on their molecular biology has been generated, there is little data on the physiology of these cultures. Different mouse strains show clear differences in behavioral and other neurobiologically relevant readouts. We have studied the physiology of early differentiation and network formation in neuronal cultures derived from three different mouse embryonic stem cell lines. We have found largely overlapping patterns with some significant differences in the timing of the functional milestones. Neurons from R1 showed the fastest development of intrinsic excitability, while E14Tg2a and J1 were slower. This was also reflected in an earlier appearance of synaptic activity in R1 cultures, while E14Tg2a and J1 were delayed by up to 2 days. In conclusion, stem cells from all backgrounds could be successfully differentiated into functioning neural networks with similar developmental patterns. Differences in the timing of specific milestones, suggest that control cell lines and time-points should be carefully chosen when investigating genetic alterations that lead to subtle deficits in neuronal function. PMID:24600351

  6. Importance of being Nernst: Synaptic activity and functional relevance in stem cell-derived neurons

    PubMed Central

    Bradford, Aaron B; McNutt, Patrick M

    2015-01-01

    Functional synaptogenesis and network emergence are signature endpoints of neurogenesis. These behaviors provide higher-order confirmation that biochemical and cellular processes necessary for neurotransmitter release, post-synaptic detection and network propagation of neuronal activity have been properly expressed and coordinated among cells. The development of synaptic neurotransmission can therefore be considered a defining property of neurons. Although dissociated primary neuron cultures readily form functioning synapses and network behaviors in vitro, continuously cultured neurogenic cell lines have historically failed to meet these criteria. Therefore, in vitro-derived neuron models that develop synaptic transmission are critically needed for a wide array of studies, including molecular neuroscience, developmental neurogenesis, disease research and neurotoxicology. Over the last decade, neurons derived from various stem cell lines have shown varying ability to develop into functionally mature neurons. In this review, we will discuss the neurogenic potential of various stem cells populations, addressing strengths and weaknesses of each, with particular attention to the emergence of functional behaviors. We will propose methods to functionally characterize new stem cell-derived neuron (SCN) platforms to improve their reliability as physiological relevant models. Finally, we will review how synaptically active SCNs can be applied to accelerate research in a variety of areas. Ultimately, emphasizing the critical importance of synaptic activity and network responses as a marker of neuronal maturation is anticipated to result in in vitro findings that better translate to efficacious clinical treatments. PMID:26240679

  7. In vitro production of functional immune cells derived from human hematopoietic stem cells

    PubMed Central

    Payuhakrit, Witchuda; Panichakul, Tasanee; Charoenphon, Natthawut; Chalermsaenyakorn, Panus; Jaovisidha, Adithep; Wongborisuth, Chokdee; Udomsangpetch, Rachanee

    2015-01-01

    Hematopoietic stem cells (HSC) from cord blood are potentially high sources for transplantation due to their low immunogenicity and the presence of the multipotent cells. These cells are capable of differentiating to produce various lineages of blood cells under specific conditions. We have enriched highly purified CD34+ cells from cord blood, determined in vitro growth of the cells in culture systems in the absence (condition A) or presence of GM-CSF and G-CSF (condition B), and determined the profile of immune cells during the period of cultivation by using flow cytometry. PhytohemagglutininA (PHA) was used as a mitogen to stimulate T lymphocytes derived from hematopoietic stem cells. GM-CSF and G-CSF prolonged the survival of the growing cells and also maintained expansion of cells in blastic stage. By day 12 of cultivation, when cell numbers peaked, various types of immune cells had appeared (CD14+ cells, CD40+HLA-DR+ cells, CD3+CD56+ cells, CD19+ cells, CD3+CD4+ cells, CD3+CD8+cells and CD3-CD56+). A significantly higher percentage of monocytes (p = 0.002) were observed under culture with GM-CSF, G-CSF when compared with culture without GM-CSF, G-CSF. In addition, T lymphocytes derived from HSC responded to 50 µg/ml of PHA. This is the first report showing the complete differentiation and proliferation of immune cells derived from CD34+ HSC under in vitro culture conditions. Lymphocytes, monocytes, dendritic cells and polymorph nuclear cells derived from HSC in vitro are unique, and thus may benefit various studies such as innate immunity and pathophysiology of immune disorders. PMID:26933404

  8. Stem cell-derived angiogenic/vasculogenic cells: possible therapies for tissue repair and tissue engineering.

    PubMed

    Zwaginga, J J; Doevendans, P

    2003-11-01

    1. The recent ability to isolate stem cells and study their specific capacity of self-renewal with the formation of different cell types has opened up exciting vistas to help the repair of damaged tissue and even the formation of new tissue. In the present review, we deal with the characteristics and sources that stem cells can be derived and cultured from. 2. We focus on the role that stem cell-derived vascular cells or endothelial progenitor cells (EPC) may play in (re)vascularization of ischaemic and engineered tissues. This so-called vasculogenesis resembles the embryological process in which 'haemangioblasts' differentiate in blood cells, as well as in primitive vessels. Although also derived from the blood-forming bone marrow, in adult life vasculogenic stem cells contribute only little to the regular vascular repair mechanisms: namely (i) angiogenesis (outgrowth of vessels from existing vessels); and (ii) arteriogenesis (monocyte-aided increase in the calibre of existing arteriolar collaterals). 3. Most attempts to increase vascular repair by stem cells involve the use of growth factors, which mobilize stem cells from bone marrow into the blood, sometimes combined with isolation and reinfusion of these cells after ex vivo expansion and differentiation into EPC. 4. Clear improved perfusion of ischaemic sites and new vasculature has been observed in vivo mostly in animal models. Specific homing or administration of these cells and regulated and quantitative expansion and (final) differentiation at these vascular (repair) sites are less studied, but are paramount for efficacy and safety. 5. In conclusion, the use of embryonic stem cells will still encounter ethical objections. Moreover, special attention and measures are needed to cope with the allogeneic barriers that these cells usually encounter. In general, the long and complicated ex vivo cultures to obtain sufficient offspring from the very small numbers of stem cells that can be obtained as starting

  9. A Non-invasive Platform for Functional Characterization of Stem-Cell-Derived Cardiomyocytes with Applications in Cardiotoxicity Testing

    PubMed Central

    Maddah, Mahnaz; Heidmann, Julia D.; Mandegar, Mohammad A.; Walker, Chase D.; Bolouki, Sara; Conklin, Bruce R.; Loewke, Kevin E.

    2015-01-01

    Summary We present a non-invasive method to characterize the function of pluripotent stem-cell-derived cardiomyocytes based on video microscopy and image analysis. The platform, called Pulse, generates automated measurements of beating frequency, beat duration, amplitude, and beat-to-beat variation based on motion analysis of phase-contrast images captured at a fast frame rate. Using Pulse, we demonstrate recapitulation of drug effects in stem-cell-derived cardiomyocytes without the use of exogenous labels and show that our platform can be used for high-throughput cardiotoxicity drug screening and studying physiologically relevant phenotypes. PMID:25801505

  10. Examining the Protective Role of ErbB2 Modulation in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Eldridge, Sandy; Mussio, Jodie; Furniss, Mike; Hamre, John; Davis, Myrtle

    2014-01-01

    Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as an in vitro model system in cardiac biology and in drug discovery (e.g., cardiotoxicity testing). Qualification of these cells for use in mechanistic investigations will require detailed evaluations of cardiomyocyte signaling pathways and cellular responses. ErbB signaling and the ligand neuregulin play critical roles in survival and functional integrity of cardiac myocytes. As such, we sought to characterize the expression and activity of the ErbB family of receptors. Antibody microarray analysis performed on cell lysates derived from maturing hiPSC-CMs detected expression of ∼570 signaling proteins. EGFR/ErbB1, HER2/ErbB2, and ErbB4, but not ErbB3 receptors, of the epidermal growth factor receptor family were confirmed by Western blot. Activation of ErbB signaling by neuregulin-1β (NRG, a natural ligand for ErbB4) and its modulation by trastuzumab (a monoclonal anti-ErbB2 antibody) and lapatinib (a small molecule ErbB2 tyrosine kinase inhibitor) were evaluated through assessing phosphorylation of AKT and Erk1/2, two major downstream kinases of ErbB signaling, using nanofluidic proteomic immunoassay. Downregulation of ErbB2 expression by siRNA silencing attenuated NRG-induced AKT and Erk1/2 phosphorylation. Activation of ErbB signaling with NRG, or inhibition with trastuzumab, alleviated or aggravated doxorubicin-induced cardiomyocyte damage, respectively, as assessed by a real-time cellular impedance analysis and ATP measurement. Collectively, these results support the expanded use of hiPSC-CMs to examine mechanisms of cardiotoxicity and support the value of using these cells in early assessments of cardiotoxicity or efficacy. PMID:25055963

  11. Differential responses of induced pluripotent stem cell-derived cardiomyocytes to anisotropic strain depends on disease status.

    PubMed

    Chun, Young Wook; Voyles, David E; Rath, Rutwik; Hofmeister, Lucas H; Boire, Timothy C; Wilcox, Henry; Lee, Jae Han; Bellan, Leon M; Hong, Charles C; Sung, Hak-Joon

    2015-11-01

    Primary dilated cardiomyopathy (DCM) is a non-ischemic heart disease with impaired pumping function of the heart. In this study, we used human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a healthy volunteer and a primary DCM patient to investigate the impact of DCM on iPSC-CMs׳ responses to different types of anisotropic strain. A bioreactor system was established that generates cardiac-mimetic forces of 150 kPa at 5% anisotropic cyclic strain and 1 Hz frequency. After confirming cardiac induction of the iPSCs, it was determined that fibronectin was favorable to other extracellular matrix protein coatings (gelatin, laminin, vitronectin) in terms of viable cell area and density, and was therefore selected as the coating for further study. When iPSC-CMs were exposed to three strain conditions (no strain, 5% static strain, and 5% cyclic strain), the static strain elicited significant induction of sarcomere components in comparison to other strain conditions. However, this induction occurred only in iPSC-CMs from a healthy volunteer ("control iPSC-CMs"), not in iPSC-CMs from the DCM patient ("DCM iPSC-CMs"). The donor type also significantly influenced gene expressions of cell-cell and cell-matrix interaction markers in response to the strain conditions. Gene expression of connexin-43 (cell-cell interaction) had a higher fold change in healthy versus diseased iPSC-CMs under static and cyclic strain, as opposed to integrins α-5 and α-10 (cell-matrix interaction). In summary, our iPSC-CM-based study to model the effects of different strain conditions suggests that intrinsic, genetic-based differences in the cardiomyocyte responses to strain may influence disease manifestation in vivo. PMID:26476764

  12. Examining the protective role of ErbB2 modulation in human-induced pluripotent stem cell-derived cardiomyocytes.

    PubMed

    Eldridge, Sandy; Guo, Liang; Mussio, Jodie; Furniss, Mike; Hamre, John; Davis, Myrtle

    2014-10-01

    Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are being used as an in vitro model system in cardiac biology and in drug discovery (e.g., cardiotoxicity testing). Qualification of these cells for use in mechanistic investigations will require detailed evaluations of cardiomyocyte signaling pathways and cellular responses. ErbB signaling and the ligand neuregulin play critical roles in survival and functional integrity of cardiac myocytes. As such, we sought to characterize the expression and activity of the ErbB family of receptors. Antibody microarray analysis performed on cell lysates derived from maturing hiPSC-CMs detected expression of ∼570 signaling proteins. EGFR/ErbB1, HER2/ErbB2, and ErbB4, but not ErbB3 receptors, of the epidermal growth factor receptor family were confirmed by Western blot. Activation of ErbB signaling by neuregulin-1β (NRG, a natural ligand for ErbB4) and its modulation by trastuzumab (a monoclonal anti-ErbB2 antibody) and lapatinib (a small molecule ErbB2 tyrosine kinase inhibitor) were evaluated through assessing phosphorylation of AKT and Erk1/2, two major downstream kinases of ErbB signaling, using nanofluidic proteomic immunoassay. Downregulation of ErbB2 expression by siRNA silencing attenuated NRG-induced AKT and Erk1/2 phosphorylation. Activation of ErbB signaling with NRG, or inhibition with trastuzumab, alleviated or aggravated doxorubicin-induced cardiomyocyte damage, respectively, as assessed by a real-time cellular impedance analysis and ATP measurement. Collectively, these results support the expanded use of hiPSC-CMs to examine mechanisms of cardiotoxicity and support the value of using these cells in early assessments of cardiotoxicity or efficacy. PMID:25055963

  13. PDX1-engineered embryonic stem cell-derived insulin producing cells regulate hyperglycemia in diabetic mice

    PubMed Central

    2012-01-01

    Background Type 1 diabetes can be treated by the transplantation of cadaveric whole pancreata or isolated pancreatic islets. However, this form of treatment is hampered by the chronic shortage of cadaveric donors. Embryonic stem (ES) cell-derived insulin producing cells (IPCs) offer a potentially novel source of unlimited cells for transplantation to treat type 1 and possibly type 2 diabetes. However, thus far, the lack of a reliable protocol for efficient differentiation of ES cells into IPCs has hindered the clinical exploitation of these cells. Methods To efficiently generate IPCs using ES cells, we have developed a double transgenic ES cell line R1Pdx1AcGFP/RIP-Luc that constitutively expresses pancreatic β-cell-specific transcription factor pancreatic and duodenal homeobox gene 1 (Pdx1) as well as rat insulin promoter (RIP) driven luciferase reporter. We have established several protocols for the reproducible differentiation of ES cells into IPCs. The differentiation of ES cells into IPCs was monitored by immunostaining as well as real-time quantitative RT-PCR for pancreatic β-cell-specific markers. Pancreatic β-cell specific RIP became transcriptionally active following the differentiation of ES cells into IPCs and induced the expression of the luciferase reporter. Glucose stimulated insulin secretion by the ES cell-derived IPCs was measured by ELISA. Further, we have investigated the therapeutic efficacy of ES cell-derived IPCs to correct hyperglycemia in syngeneic streptozotocin (STZ)-treated diabetic mice. The long term fate of the transplanted IPCs co-expressing luciferase in syngeneic STZ-induced diabetic mice was monitored by real time noninvasive in vivo bioluminescence imaging (BLI). Results We have recently demonstrated that spontaneous in vivo differentiation of R1Pdx1AcGFP/RIP-Luc ES cell-derived pancreatic endoderm-like cells (PELCs) into IPCs corrects hyperglycemia in diabetic mice. Here, we investigated whether R1Pdx1AcGFP/RIP-Luc ES cells

  14. Inhibition of pluripotent stem cell-derived teratoma formation by small molecules.

    PubMed

    Lee, Mi-Ok; Moon, Sung Hwan; Jeong, Ho-Chang; Yi, Ji-Yeon; Lee, Tae-Hee; Shim, Sung Han; Rhee, Yong-Hee; Lee, Sang-Hun; Oh, Seok-Jeong; Lee, Moo-Yeol; Han, Min-Joon; Cho, Yee Sook; Chung, Hyung-Min; Kim, Kwang-Soo; Cha, Hyuk-Jin

    2013-08-27

    The future of safe cell-based therapy rests on overcoming teratoma/tumor formation, in particular when using human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Because the presence of a few remaining undifferentiated hPSCs can cause undesirable teratomas after transplantation, complete removal of these cells with no/minimal damage to differentiated cells is a prerequisite for clinical application of hPSC-based therapy. Having identified a unique hESC signature of pro- and antiapoptotic gene expression profile, we hypothesized that targeting hPSC-specific antiapoptotic factor(s) (i.e., survivin or Bcl10) represents an efficient strategy to selectively eliminate pluripotent cells with teratoma potential. Here we report the successful identification of small molecules that can effectively inhibit these antiapoptotic factors, leading to selective and efficient removal of pluripotent stem cells through apoptotic cell death. In particular, a single treatment of hESC-derived mixed population with chemical inhibitors of survivin (e.g., quercetin or YM155) induced selective and complete cell death of undifferentiated hPSCs. In contrast, differentiated cell types (e.g., dopamine neurons and smooth-muscle cells) derived from hPSCs survived well and maintained their functionality. We found that quercetin-induced selective cell death is caused by mitochondrial accumulation of p53 and is sufficient to prevent teratoma formation after transplantation of hESC- or hiPSC-derived cells. Taken together, these results provide the "proof of concept" that small-molecule targeting of hPSC-specific antiapoptotic pathway(s) is a viable strategy to prevent tumor formation by selectively eliminating remaining undifferentiated pluripotent cells for safe hPSC-based therapy. PMID:23918355

  15. Human pluripotent stem cell-derived limbal epithelial stem cells on bioengineered matrices for corneal reconstruction.

    PubMed

    Mikhailova, Alexandra; Ilmarinen, Tanja; Ratnayake, Anjula; Petrovski, Goran; Uusitalo, Hannu; Skottman, Heli; Rafat, Mehrdad

    2016-05-01

    Corneal epithelium is renewed by limbal epithelial stem cells (LESCs), a type of tissue-specific stem cells located in the limbal palisades of Vogt at the corneo-scleral junction. Acute trauma or inflammatory disorders of the ocular surface can destroy these stem cells, leading to limbal stem cell deficiency (LSCD) - a painful and vision-threatening condition. Treating these disorders is often challenging and complex, especially in bilateral cases with extensive damage. Human pluripotent stem cells (hPSCs) provide new opportunities for corneal reconstruction using cell-based therapy. Here, we investigated the use of hPSC-derived LESC-like cells on bioengineered collagen matrices in serum-free conditions, aiming for clinical applications to reconstruct the corneal epithelium and partially replace the damaged stroma. Differentiation of hPSCs towards LESC-like cells was directed using small-molecule induction followed by maturation in corneal epithelium culture medium. After four to five weeks of culture, differentiated cells were seeded onto bioengineered matrices fabricated as transparent membranes of uniform thickness, using medical-grade porcine collagen type I and a hybrid cross-linking technology. The bioengineered matrices were fully transparent, with high water content and swelling capacity, and parallel lamellar microstructure. Cell proliferation of hPSC-LESCs was significantly higher on bioengineered matrices than on collagen-coated control wells after two weeks of culture, and LESC markers p63 and cytokeratin 15, along with proliferation marker Ki67 were expressed even after 30 days in culture. Overall, hPSC-LESCs retained their capacity to self-renew and proliferate, but were also able to terminally differentiate upon stimulation, as suggested by protein expression of cytokeratins 3 and 12. We propose the use of bioengineered collagen matrices as carriers for the clinically-relevant hPSC-derived LESC-like cells, as a novel tissue engineering approach for

  16. Mesenchymal stem cells and cardiac repair

    PubMed Central

    Nesselmann, Catharina; Ma, Nan; Bieback, Karen; Wagner, Wolfgang; Ho, Anthony; Konttinen, Yrjö T; Zhang, Hao; Hinescu, Mihail E; Steinhoff, Gustav

    2008-01-01

    Accumulating clinical and experimental evidence indicates that mesenchymal stem cells (MSCs) are promising cell types in the treatment of cardiac dysfunction. They may trigger production of reparative growth factors, replace damaged cells and create an environment that favours endogenous cardiac repair. However, identifying mechanisms which regulate the role of MSCs in cardiac repair is still at work. To achieve the maximal clinical benefits, ex vivo manipulation can further enhance MSC therapeutic potential. This review focuses on the mechanism of MSCs in cardiac repair, with emphasis on ex vivo manipulation. PMID:18684237

  17. Characterization of human-induced pluripotent stem cell-derived cardiomyocytes: bioenergetics and utilization in safety screening.

    PubMed

    Rana, Payal; Anson, Blake; Engle, Sandra; Will, Yvonne

    2012-11-01

    Cardiotoxicity remains the number one reason for drug withdrawal from the market, and Food and Drug Administration issued black box warnings, thus demonstrating the need for more predictive preclinical safety screening, especially early in the drug discovery process when much chemical substrate is available. Whereas human-ether-a-go-go related gene screening has become routine to mitigate proarrhythmic risk, the development of in vitro assays predicting additional on- and off-target biochemical toxicities will benefit from cellular models exhibiting true cardiomyocyte characteristics such as native tissue-like mitochondrial activity. Human stem cell-derived tissue cells may provide such a model. This hypothesis was tested using a combination of flux analysis, gene and protein expression, and toxicity-profiling techniques to characterize mitochondrial function in induced pluripotent stem cell (iPSC) derived human cardiomyocytes in the presence of differing carbon sources over extended periods in cell culture. Functional analyses demonstrate that iPSC-derived cardiomyocytes are (1) capable of utilizing anaerobic or aerobic respiration depending upon the available carbon substrate and (2) bioenergetically closest to adult heart tissue cells when cultured in galactose or galactose supplemented with fatty acids. We utilized this model to test a variety of kinase inhibitors with known clinical cardiac liabilities for their potential toxicity toward these cells. We found that the kinase inhibitors showed a dose-dependent toxicity to iPSC cardiomyocytes grown in galactose and that oxygen consumption rates were significantly more affected than adenosine triphosphate production. Sorafenib was found to have the most effect, followed by sunitinib, dasatinib, imatinib, lapatinib, and nioltinib. PMID:22843568

  18. Long Term Liver Engraftment of Functional Hepatocytes Obtained from Germline Cell-Derived Pluripotent Stem Cells

    PubMed Central

    Fagoonee, Sharmila; Famulari, Elvira Smeralda; Silengo, Lorenzo; Tolosano, Emanuela; Altruda, Fiorella

    2015-01-01

    One of the major hurdles in liver gene and cell therapy is availability of ex vivo-expanded hepatocytes. Pluripotent stem cells are an attractive alternative. Here, we show that hepatocyte precursors can be isolated from male germline cell-derived pluripotent stem cells (GPSCs) using the hepatoblast marker, Liv2, and induced to differentiate into hepatocytes in vitro. These cells expressed hepatic-specific genes and were functional as demonstrated by their ability to secrete albumin and produce urea. When transplanted in the liver parenchyma of partially hepatectomised mice, Liv2-sorted cells showed regional and heterogeneous engraftment in the injected lobe. Moreover, approximately 50% of Y chromosome-positive, GPSC-derived cells were found in the female livers, in the region of engraftment, even one month after cell injection. This is the first study showing that Liv2-sorted GPSCs-derived hepatocytes can undergo long lasting engraftment in the mouse liver. Thus, GPSCs might offer promise for regenerative medicine. PMID:26323094

  19. Paracrine Effect of Mesenchymal Stem Cells Derived from Human Adipose Tissue in Bone Regeneration

    PubMed Central

    Linero, Itali; Chaparro, Orlando

    2014-01-01

    Mesenchymal stem cell (MSC) transplantation has proved to be a promising strategy in cell therapy and regenerative medicine. Although their mechanism of action is not completely clear, it has been suggested that their therapeutic activity may be mediated by a paracrine effect. The main goal of this study was to evaluate by radiographic, morphometric and histological analysis the ability of mesenchymal stem cells derived from human adipose tissue (Ad-MSC) and their conditioned medium (CM), to repair surgical bone lesions using an in vivo model (rabbit mandibles). The results demonstrated that both, Ad-MSC and CM, induce bone regeneration in surgically created lesions in rabbit's jaws, suggesting that Ad-MSC improve the process of bone regeneration mainly by releasing paracrine factors. The evidence of the paracrine effect of MSC on bone regeneration has a major impact on regenerative medicine, and the use of their CM can address some issues and difficulties related to cell transplants. In particular, CM can be easily stored and transported, and is easier to handle by medical personnel during clinical procedures. PMID:25198551

  20. Functional Kidney Bioengineering with Pluripotent Stem-Cell-Derived Renal Progenitor Cells and Decellularized Kidney Scaffolds.

    PubMed

    Du, Chan; Narayanan, Karthikeyan; Leong, Meng Fatt; Ibrahim, Mohammed Shahrudin; Chua, Ying Ping; Khoo, Vanessa Mei Hui; Wan, Andrew C A

    2016-08-01

    Recent advances in developmental biology and stem cell technology have led to the engineering of functional organs in a dish. However, the limited size of these organoids and absence of a large circulatory system poses limits to its clinical translation. To overcome these issues, decellularized whole kidney scaffolds with native microstructure and extracellular matrix (ECM) are employed for kidney bioengineering, using human-induced pluripotent-stem-cell-derived renal progenitor cells and endothelial cells. To demonstrate ECM-guided cellular assembly, the present work is focused on generating the functional unit of the kidney, the glomerulus. In the repopulated organ, the presence of endothelial cells broadly upregulates the expression level of genes related to renal development. When the cellularized native scaffolds are implanted in SCID mice, glomeruli assembly can be achieved by co-culture of the renal progenitors and endothelial cells. These individual glomerular units are shown to be functional in the context of the whole organ using a simulated bio-reactor set-up with urea and creatinine excretion and albumin reabsorption. Our results indicate that the repopulation of decellularized native kidney using clinically relevant, expandable patient-specific renal progenitors and endothelial cells may be a viable approach for the generation of a functional whole kidney. PMID:27294565

  1. Engineered Microenvironments for the Maturation and Observation of Human Embryonic Stem Cell Derived Cardiomyocytes

    NASA Astrophysics Data System (ADS)

    Salick, Max R.

    The human heart is a dynamic system that undergoes substantial changes as it develops and adapts to the body's growing needs. To better understand the physiology of the heart, researchers have begun to produce immature heart muscle cells, or cardiomyocytes, from pluripotent stem cell sources with remarkable efficiency. These stem cell-derived cardiomyocytes hold great potential in the understanding and treatment of heart disease; however, even after prolonged culture, these cells continue to exhibit an immature phenotype, as indicated by poor sarcomere organization and calcium handling, among other features. The lack of maturation that is observed in these cardiomyocytes greatly limits their applicability towards drug screening, disease modeling, and cell therapy applications. The mechanical environment surrounding a cell has been repeatedly shown to have a large impact on that cell's behavior. For this reason, we have implemented micropatterning methods to mimic the level of alignment that occurs in the heart in vivo in order to study how this alignment may help the cells to produce a more mature sarcomere phenotype. It was discovered that the level of sarcomere organization of a cardiomyocyte can be strongly influenced by the micropattern lane geometry on which it adheres. Steps were taken to optimize this micropattern platform, and studies of protein organization, gene expression, and myofibrillogenesis were conducted. Additionally, a set of programs was developed to provide quantitative analysis of the level of sarcomere organization, as well as to assist with several other tissue engineering applications.

  2. Engraftment of embryonic stem cell-derived myogenic progenitors in a dominant model of muscular dystrophy

    PubMed Central

    Darabi, Radbod; Baik, June; Clee, Mark; Kyba, Michael; Tupler, Rossella; Perlingeiro, Rita C.R.

    2009-01-01

    Muscular dystrophies (MD) consist of a genetically heterogeneous group of disorders, recessive or dominant, characterized by progressive skeletal muscle weakening. To date, no effective treatment is available. Experimental strategies pursuing muscle regeneration through the transplantation of stem cell preparations have brought hope to patients affected by this disorder. Efficacy has been demonstrated in recessive MD models through contribution of wild-type nuclei to the muscle fiber heterokaryon, however to date, there has been no study investigating the efficacy of a cell therapy in a dominant model of MD. We have recently demonstrated that Pax3-induced embryonic stem (ES) cell- derived myogenic progenitors are able to engraft and improve muscle function in mdx mice, a recessive mouse model for Duchenne MD. To assess whether this therapeutic effect can be extended to a dominant type of muscle disorder, here we transplanted these cells into FRG1 transgenic mice, a dominant model that has been associated with Facioscapulohumeral muscular dystrophy. Our results show that Pax3-induced ES-derived myogenic progenitors are capable of significant engraftment after intramuscular or systemic transplantation into Frg1 mice. Analyses of contractile parameters revealed functional improvement in treated muscles of male mice, but not females, which are less severely affected. This study is the first to use Frg1 transgenic mice to assess muscle regeneration as well as to support the use of a cell-based therapy for autosomal dominant types of MD. PMID:19682990

  3. G-protein Coupled Receptor Signaling in Pluripotent Stem Cell-derived Cardiovascular Cells: Implications for Disease Modeling

    PubMed Central

    Dolatshad, Nazanin F.; Hellen, Nicola; Jabbour, Richard J.; Harding, Sian E.; Földes, Gabor

    2015-01-01

    Human pluripotent stem cell derivatives show promise as an in vitro platform to study a range of human cardiovascular diseases. A better understanding of the biology of stem cells and their cardiovascular derivatives will help to understand the strengths and limitations of this new model system. G-protein coupled receptors (GPCRs) are key regulators of stem cell maintenance and differentiation and have an important role in cardiovascular cell signaling. In this review, we will therefore describe the state of knowledge concerning the regulatory role of GPCRs in both the generation and function of pluripotent stem cell derived-cardiomyocytes, -endothelial, and -vascular smooth muscle cells. We will consider how far the in vitro disease models recapitulate authentic GPCR signaling and provide a useful basis for discovery of disease mechanisms or design of therapeutic strategies. PMID:26697426

  4. Cardiac Regeneration Using Pluripotent Stem Cells – Progression to Large Animal Models

    PubMed Central

    Chong, James J.H.; Murry, Charles E.

    2014-01-01

    Pluripotent stem cells (PSCs) have indisputable cardiomyogenic potential and therefore have been intensively investigated as a potential cardiac regenerative therapy. Current directed differentiation protocols are able to produce high yields of cardiomyocytes from PSCs and studies in small animal models of cardiovascular disease have proven sustained engraftment and functional efficacy. Therefore, the time is ripe for cardiac regenerative therapies using PSC derivatives to be tested in large animal models that more closely resemble the hearts of humans. In this review, we discuss the results of our recent study using human embryonic stem cell derived cardiomyocytes (hESC-CM) in a non-human primate model of ischemic cardiac injury. Large scale remuscularization, electromechanical coupling and short-term arrhythmias demonstrated by our hESC-CM grafts are discussed in the context of other studies using Adult Stem Cells for cardiac regeneration. PMID:25087896

  5. In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons.

    PubMed

    Park, Chang-Hwan; Minn, Yang-Ki; Lee, Ji-Yeon; Choi, Dong Ho; Chang, Mi-Yoon; Shim, Jae-Won; Ko, Ji-Yun; Koh, Hyun-Chul; Kang, Min Jeong; Kang, Jin Sun; Rhie, Duck-Joo; Lee, Yong-Sung; Son, Hyeon; Moon, Shin Yong; Kim, Kwang-Soo; Lee, Sang-Hun

    2005-03-01

    Human embryonic stem (hES) cells, due to their capacity of multipotency and self-renewal, may serve as a valuable experimental tool for human developmental biology and may provide an unlimited cell source for cell replacement therapy. The purpose of this study was to assess the developmental potential of hES cells to replace the selectively lost midbrain dopamine (DA) neurons in Parkinson's disease. Here, we report the development of an in vitro differentiation protocol to derive an enriched population of midbrain DA neurons from hES cells. Neural induction of hES cells co-cultured with stromal cells, followed by expansion of the resulting neural precursor cells, efficiently generated DA neurons with concomitant expression of transcriptional factors related to midbrain DA development, such as Pax2, En1 (Engrailed-1), Nurr1, and Lmx1b. Using our procedure, the majority of differentiated hES cells (> 95%) contained neuronal or neural precursor markers and a high percentage (> 40%) of TuJ1+ neurons was tyrosine hydroxylase (TH)+, while none of them expressed the undifferentiated ES cell marker, Oct 3/4. Furthermore, hES cell-derived DA neurons demonstrated functionality in vitro, releasing DA in response to KCl-induced depolarization and reuptake of DA. Finally, transplantation of hES-derived DA neurons into the striatum of hemi-parkinsonian rats failed to result in improvement of their behavioral deficits as determined by amphetamine-induced rotation and step-adjustment. Immunohistochemical analyses of grafted brains revealed that abundant hES-derived cells (human nuclei+ cells) survived in the grafts, but none of them were TH+. Therefore, unlike those from mouse ES cells, hES cell-derived DA neurons either do not survive or their DA phenotype is unstable when grafted into rodent brains. PMID:15715675

  6. Insulin-producing cells derived from stem cells: recent progress and future directions.

    PubMed

    Santana, A; Enseñat-Waser, R; Arribas, María Isabel; Reig, J A; Roche, E

    2006-01-01

    Type 1 diabetes is characterized by the selective destruction of pancreatic beta-cells caused by an autoimmune attack. Type 2 diabetes is a more complex pathology which, in addition to beta-cell loss caused by apoptotic programs, includes beta-cell dedifferentiation and peripheric insulin resistance. beta-Cells are responsible for insulin production, storage and secretion in accordance to the demanding concentrations of glucose and fatty acids. The absence of insulin results in death and therefore diabetic patients require daily injections of the hormone for survival. However, they cannot avoid the appearance of secondary complications affecting the peripheral nerves as well as the eyes, kidneys and cardiovascular system. These afflictions are caused by the fact that external insulin injection does not mimic the tight control that pancreatic-derived insulin secretion exerts on the body's glycemia. Restoration of damaged beta-cells by transplantation from exogenous sources or by endocrine pancreas regeneration would be ideal therapeutic options. In this context, stem cells of both embryonic and adult origin (including beta-cell/islet progenitors) offer some interesting alternatives, taking into account the recent data indicating that these cells could be the building blocks from which insulin secreting cells could be generated in vitro under appropriate culture conditions. Although in many cases insulin-producing cells derived from stem cells have been shown to reverse experimentally induced diabetes in animal models, several concerns need to be solved before finding a definite medical application. These refer mainly to the obtainment of a cell population as similar as possible to pancreatic beta-cells, and to the problems related with the immune compatibility and tumor formation. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells, and the main problems that hamper the

  7. Insulin - producing cells derived from stem cells: recent progress and future directions

    PubMed Central

    Santana, A; Enseñat - Waser, R; Arribas, Maria Isabel; Reig, J A; Roche, E

    2006-01-01

    Type 1 diabetes is characterized by the selective destruction of pancreatic β-cells caused by an autoimmune attack. Type 2 diabetes is a more complex pathology which, in addition to β-cell loss caused by apoptotic programs, includes β-cell dedifferentiation and peripheric insulin resistance. β-Cells are responsible for insulin production, storage and secretion in accordance to the demanding concentrations of glucose and fatty acids. The absence of insulin results in death and therefore diabetic patients require daily injections of the hormone for survival. However, they cannot avoid the appearance of secondary complications affecting the peripheral nerves as well as the eyes, kidneys and cardiovascular system. These afflictions are caused by the fact that external insulin injection does not mimic the tight control that pancreaticderived insulin secretion exerts on the body’s glycemia. Restoration of damaged β-cells by transplantation from exogenous sources or by endocrine pancreas regeneration would be ideal therapeutic options. In this context, stem cells of both embryonic and adult origin (including β-cell/islet progenitors) offer some interesting alternatives, taking into account the recent data indicating that these cells could be the building blocks from which insulin secreting cells could be generated in vitro under appropriate culture conditions. Although in many cases insulin-producing cells derived from stem cells have been shown to reverse experimentally induced diabetes in animal models, several concerns need to be solved before finding a definite medical application. These refer mainly to the obtainment of a cell population as similar as possible to pancreatic β-cells, and to the problems related with the immune compatibility and tumor formation. This review will summarize the different approaches that have been used to obtain insulin-producing cells from embryonic and adult stem cells, and the main problems that hamper the clinical

  8. Pathogen Sensing Pathways in Human Embryonic Stem Cell Derived-Endothelial Cells: Role of NOD1 Receptors

    PubMed Central

    Reed, Daniel M.; Foldes, Gabor; Gatheral, Timothy; Paschalaki, Koralia E.; Lendvai, Zsuzsanna; Bagyura, Zsolt; Nemeth, Tamas; Skopal, Judit; Merkely, Bela; Telcian, Aurica G.; Gogsadze, Leila; Edwards, Michael R.; Gough, Peter J.; Bertin, John; Johnston, Sebastian L.; Harding, Sian E.; Mitchell, Jane A.

    2014-01-01

    Human embryonic stem cell-derived endothelial cells (hESC-EC), as well as other stem cell derived endothelial cells, have a range of applications in cardiovascular research and disease treatment. Endothelial cells sense Gram-negative bacteria via the pattern recognition receptors (PRR) Toll-like receptor (TLR)-4 and nucleotide-binding oligomerisation domain-containing protein (NOD)-1. These pathways are important in terms of sensing infection, but TLR4 is also associated with vascular inflammation and atherosclerosis. Here, we have compared TLR4 and NOD1 responses in hESC-EC with those of endothelial cells derived from other stem cells and with human umbilical vein endothelial cells (HUVEC). HUVEC, endothelial cells derived from blood progenitors (blood outgrowth endothelial cells; BOEC), and from induced pluripotent stem cells all displayed both a TLR4 and NOD1 response. However, hESC-EC had no TLR4 function, but did have functional NOD1 receptors. In vivo conditioning in nude rats did not confer TLR4 expression in hESC-EC. Despite having no TLR4 function, hESC-EC sensed Gram-negative bacteria, a response that was found to be mediated by NOD1 and the associated RIP2 signalling pathways. Thus, hESC-EC are TLR4 deficient but respond to bacteria via NOD1. This data suggests that hESC-EC may be protected from unwanted TLR4-mediated vascular inflammation, thus offering a potential therapeutic advantage. PMID:24690886

  9. Induced pluripotent stem cell-derived neuron as a human model for testing environmentally induced developmental neurotoxicity

    EPA Science Inventory

    Induced pluripotent stem cell-derived neurons as a human model for testing environmentally induced developmental neurotoxicity Ingrid L. Druwe1, Timothy J. Shafer2, Kathleen Wallace2, Pablo Valdivia3 ,and William R. Mundy2. 1University of North Carolina, Curriculum in Toxicology...

  10. Regeneration of infarcted myocardium with resveratrol-modified cardiac stem cells

    PubMed Central

    Gorbunov, Nikolai; Petrovski, Goran; Gurusamy, Narasimman; Ray, Diptarka; Kim, Do Han; Das, Dipak K

    2012-01-01

    Abstract The major problem in stem cell therapy includes viability and engraftment efficacy of stem cells after transplantation. Indeed, the vast majority of host-transfused cells do not survive beyond 24–72 hrs. To increase the survival and engraftment of implanted cardiac stem cells in the host, we developed a technique of treating these cells with resveratrol, and tested it in a rat model of left anterior descending (LAD) occlusion. Multi-potent clonogenic cardiac stem cells isolated from rat heart and stably transfected with EGFP were pre-treated with 2.5 μM resveratrol for 60 min. Rats were anaesthetized, hearts opened and the LAD occluded to induce heart attack. One week later, the cardiac reduced environment was confirmed in resveratrol treated rat hearts by the enhanced expression of nuclear factor-E2-related factor-2 (Nrf2) and redox effector factor-1 (Ref-1). M-mode echocardiography after stem cell therapy, showed improvement in cardiac function (left ventricular ejection fraction, fractional shortening and cardiac output) in both, the treated and control group after 7 days, but only resveratrol-modified stem cell group revealed improvement in cardiac function at the end of 1, 2 and 4 months time. The improvement of cardiac function was accompanied by enhanced stem cell survival and engraftment as demonstrated by the expression of cell proliferation marker Ki67 and differentiation of stem cells towards the regeneration of the myocardium as demonstrated by the expression of EGFP up to 4 months after LAD occlusion in the resveratrol-treated stem cell group. Expression of stromal cell-derived factor and myosin conclusively demonstrated homing of stem cells in the infarcted myocardium, its regeneration leading to improvement of cardiac function. PMID:21352470

  11. Human pluripotent stem cell-derived mesenchymal stem cells prevent allergic airway inflammation in mice.

    PubMed

    Sun, Yue-Qi; Deng, Meng-Xia; He, Jia; Zeng, Qing-Xiang; Wen, Weiping; Wong, David S H; Tse, Hung-Fat; Xu, Geng; Lian, Qizhou; Shi, Jianbo; Fu, Qing-Ling

    2012-12-01

    We previously found that mesenchymal stem cells (MSCs) derived from human-induced pluripotent stem cells (iPSCs) exerted immunomodulatory effects on Th2-mediated allergic rhinitis in vitro. However, their contribution to the asthma and allergic rhinitis in animal models remains unclear. In this study, we developed a mouse model of ovalbumin (OVA)-induced allergic inflammation in both the upper and lower airways and evaluated the effects of the systemic administration of human iPSC-MSCs and bone marrow-derived MSCs (BM-MSCs) on allergic inflammation. Our results showed that treatments with both the iPSC-MSCs and BM-MSCs before the challenge phase protected the animals from the majority of allergy-specific pathological changes. This protection included an inhibition of inflammatory cell infiltration and mucus production in the lung, a reduction in eosinophil infiltration in the nose, and a decrease in inflammatory cell infiltration in both the bronchoalveolar and nasal lavage fluids. In addition, treatment with iPSC-MSCs or BM-MSCs before the challenge phase resulted in reduced serum levels of Th2 immunoglobulins (e.g., IgE) and decreased levels of Th2 cytokines including interleukin (IL)-4, IL-5, or IL-13 in the bronchoalveolar and/or nasal lavage fluids. Similar therapeutic effects were observed when the animals were pretreated with human iPSC-MSCs before the sensitization phase. These data suggest that iPSC-MSCs may be used as an alternative strategy to adult MSCs in the treatment of asthma and allergic rhinitis. PMID:22987325

  12. Stem cell sources for cardiac regeneration.

    PubMed

    Roccio, M; Goumans, M J; Sluijter, J P G; Doevendans, P A

    2008-03-01

    Cell-based cardiac repair has the ambitious aim to replace the malfunctioning cardiac muscle developed after myocardial infarction, with new contractile cardiomyocytes and vessels. Different stem cell populations have been intensively studied in the last decade as a potential source of new cardiomyocytes to ameliorate the injured myocardium, compensate for the loss of ventricular mass and contractility and eventually restore cardiac function. An array of cell types has been explored in this respect, including skeletal muscle, bone marrow derived stem cells, embryonic stem cells (ESC) and more recently cardiac progenitor cells. The best-studied cell types are mouse and human ESC cells, which have undisputedly been demonstrated to differentiate into cardiomyocyte and vascular lineages and have been of great help to understand the differentiation process of pluripotent cells. However, due to their immunogenicity, risk of tumor development and the ethical challenge arising from their embryonic origin, they do not provide a suitable cell source for a regenerative therapy approach. A better option, overcoming ethical and allogenicity problems, seems to be provided by bone marrow derived cells and by the recently identified cardiac precursors. This report will overview current knowledge on these different cell types and their application in cardiac regeneration and address issues like implementation of delivery methods, including tissue engineering approaches that need to be developed alongside. PMID:18427385

  13. Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes

    PubMed Central

    Maillet, Agnes; Tan, Kim; Chai, Xiaoran; Sadananda, Singh N.; Mehta, Ashish; Ooi, Jolene; Hayden, Michael R.; Pouladi, Mahmoud A.; Ghosh, Sujoy; Shim, Winston; Brunham, Liam R.

    2016-01-01

    Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response. PMID:27142468

  14. Modulating Innate Immunity Improves Hepatitis C Virus Infection and Replication in Stem Cell-Derived Hepatocytes

    PubMed Central

    Zhou, Xiaoling; Sun, Pingnan; Lucendo-Villarin, Baltasar; Angus, Allan G.N.; Szkolnicka, Dagmara; Cameron, Kate; Farnworth, Sarah L.; Patel, Arvind H.; Hay, David C.

    2014-01-01

    Summary In this study, human embryonic stem cell-derived hepatocytes (hESC-Heps) were investigated for their ability to support hepatitis C virus (HCV) infection and replication. hESC-Heps were capable of supporting the full viral life cycle, including the release of infectious virions. Although supportive, hESC-Hep viral infection levels were not as great as those observed in Huh7 cells. We reasoned that innate immune responses in hESC-Heps may lead to the low level of infection and replication. Upon further investigation, we identified a strong type III interferon response in hESC-Heps that was triggered by HCV. Interestingly, specific inhibition of the JAK/STAT signaling pathway led to an increase in HCV infection and replication in hESC-Heps. Of note, the interferon response was not evident in Huh7 cells. In summary, we have established a robust cell-based system that allows the in-depth study of virus-host interactions in vitro. PMID:25068132

  15. Isolation and Mechanical Measurements of Myofibrils from Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    PubMed

    Pioner, Josè Manuel; Racca, Alice W; Klaiman, Jordan M; Yang, Kai-Chun; Guan, Xuan; Pabon, Lil; Muskheli, Veronica; Zaunbrecher, Rebecca; Macadangdang, Jesse; Jeong, Mark Y; Mack, David L; Childers, Martin K; Kim, Deok-Ho; Tesi, Chiara; Poggesi, Corrado; Murry, Charles E; Regnier, Michael

    2016-06-14

    Tension production and contractile properties are poorly characterized aspects of excitation-contraction coupling of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Previous approaches have been limited due to the small size and structural immaturity of early-stage hiPSC-CMs. We developed a substrate nanopatterning approach to produce hiPSC-CMs in culture with adult-like dimensions, T-tubule-like structures, and aligned myofibrils. We then isolated myofibrils from hiPSC-CMs and measured the tension and kinetics of activation and relaxation using a custom-built apparatus with fast solution switching. The contractile properties and ultrastructure of myofibrils more closely resembled human fetal myofibrils of similar gestational age than adult preparations. We also demonstrated the ability to study the development of contractile dysfunction of myofibrils from a patient-derived hiPSC-CM cell line carrying the familial cardiomyopathy MYH7 mutation (E848G). These methods can bring new insights to understanding cardiomyocyte maturation and developmental mechanical dysfunction of hiPSC-CMs with cardiomyopathic mutations. PMID:27161364

  16. Simvastatin modulates mouse embryonic stem cell-derived chondrogenesis in vitro.

    PubMed

    Kramer, J; Bartsch, M; Krug, D; Klinger, M; Nitschke, M; Rohwedel, J

    2012-10-01

    It has been studied in detail that cellular differentiation during chondrogenesis can be recapitulated in vitro by differentiation of embryonic stem (ES) cells as embryoid bodies (EBs). We here used this model system of cartilage development to analyze the effect of simvastatin, a potentially embryotoxic substance. Statins are a group of drugs used to treat hypercholesterolaemia. We found that simvastatin activated cartilage nodule formation during EB differentiation. Extended application of simvastatin resulted in enhanced expression of cartilage marker molecules and prolonged persistence of cartilage nodules. Expression of collagen type II was upregulated during simvastatin-induced chondrogenic ES cell differentiation as demonstrated by quantitative real time PCR. However, immunostaining for cartilage marker molecules revealed that cartilage nodules within simvastatin-treated EBs were defective, bearing cavities of cell loss. Furthermore, caspase activity was reduced in comparison to untreated controls indicating reduced apoptosis. Taken together, we may speculate that simvastatin prolongs survival of chondrocytes and disrupts cellular integrity of cartilage nodules during EB development by affecting apoptotic mechanisms. The study underlines that ES cell-derived EBs are a useful in vitro model to screen substances for their embryotoxic and teratogenic potential. PMID:22771337

  17. Tolerance of human embryonic stem cell derived islet progenitor cells to vitrification-relevant solutions.

    PubMed

    Lahmy, Reyhaneh; Bolyukh, Vladimir F; Castilla, Sergio Mora; Laurent, Louise C; Katkov, Igor I; Itkin-Ansari, Pamela

    2015-06-01

    We have previously shown that human embryonic stem cell derived islet progenitors (hESC-IPs), encapsulated inside an immunoprotective device, mature in vivo and ameliorate diabetes in mice. The ability to cryopreserve hESC-IPs preloaded in these devices would enhance consistency and portability, but traditional 'slow freezing' methods did not work well for cells encapsulated in the device. Vitrification is an attractive alternative cryopreservation approach. To assess the tolerance of hESC-IPs to vitrification relevant conditions, we here are reporting cell survival following excursions in tonicity, exposure to fifteen 40% v/v combinations of 4 cryoprotectants, and varied methods for addition and elution. We find that 78% survival is achieved using a protocol in which cells are abruptly (in one step) exposed to a solution containing 10% v/v each dimethyl sulfoxide, propylene glycol, ethylene glycol, and glycerol on ice, and eluted step-wise with DPBS+0.5M sucrose at 37°C. Importantly, the hESC-IPs also maintain expression of the critical islet progenitor markers PDX-1, NKX6.1, NGN3 and NEURO-D1. Thus, hESC-IPs exhibit robust tolerance to exposure to vitrification solutions in relevant conditions. PMID:25817378

  18. Automated Electrophysiological and Pharmacological Evaluation of Human Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Rajamohan, Divya; Kalra, Spandan; Duc Hoang, Minh; George, Vinoj; Staniforth, Andrew; Russell, Hugh; Yang, Xuebin

    2016-01-01

    Automated planar patch clamp systems are widely used in drug evaluation studies because of their ability to provide accurate, reliable, and reproducible data in a high-throughput manner. Typically, CHO and HEK tumorigenic cell lines overexpressing single ion channels are used since they can be harvested as high-density, homogenous, single-cell suspensions. While human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are physiologically more relevant, these cells are fragile, have complex culture requirements, are inherently heterogeneous, and are expensive to produce, which has restricted their use on automated patch clamp (APC) devices. Here, we used high efficiency differentiation protocols to produce cardiomyocytes from six different hPSC lines for analysis on the Patchliner (Nanion Technologies GmbH) APC platform. We developed a two-step cell preparation protocol that yielded cell catch rates and whole-cell breakthroughs of ∼80%, with ∼40% of these cells allowing electrical activity to be recorded. The protocol permitted formation of long-lasting (>15 min), high quality seals (>2 GΩ) in both voltage- and current-clamp modes. This enabled density of sodium, calcium, and potassium currents to be evaluated, along with dose–response curves to their respective channel inhibitors, tetrodotoxin, nifedipine, and E-4031. Thus, we show the feasibility of using the Patchliner platform for automated evaluation of the electrophysiology and pharmacology of hPSC-CMs, which will enable considerable increase in throughput for reliable and efficient drug evaluation. PMID:26906236

  19. Efficient Generation of Human Embryonic Stem Cell-Derived Corneal Endothelial Cells by Directed Differentiation

    PubMed Central

    McCabe, Kathryn L.; Kunzevitzky, Noelia J.; Chiswell, Brian P.; Xia, Xin; Goldberg, Jeffrey L.; Lanza, Robert

    2015-01-01

    Aim To generate human embryonic stem cell derived corneal endothelial cells (hESC-CECs) for transplantation in patients with corneal endothelial dystrophies. Materials and Methods Feeder-free hESC-CECs were generated by a directed differentiation protocol. hESC-CECs were characterized by morphology, expression of corneal endothelial markers, and microarray analysis of gene expression. Results hESC-CECs were nearly identical morphologically to primary human corneal endothelial cells, expressed Zona Occludens 1 (ZO-1) and Na+/K+ATPaseα1 (ATPA1) on the apical surface in monolayer culture, and produced the key proteins of Descemet’s membrane, Collagen VIIIα1 and VIIIα2 (COL8A1 and 8A2). Quantitative PCR analysis revealed expression of all corneal endothelial pump transcripts. hESC-CECs were 96% similar to primary human adult CECs by microarray analysis. Conclusion hESC-CECs are morphologically similar, express corneal endothelial cell markers and express a nearly identical complement of genes compared to human adult corneal endothelial cells. hESC-CECs may be a suitable alternative to donor-derived corneal endothelium. PMID:26689688

  20. Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling.

    PubMed

    Matsumoto, Takuya; Fujimori, Koki; Andoh-Noda, Tomoko; Ando, Takayuki; Kuzumaki, Naoko; Toyoshima, Manabu; Tada, Hirobumi; Imaizumi, Kent; Ishikawa, Mitsuru; Yamaguchi, Ryo; Isoda, Miho; Zhou, Zhi; Sato, Shigeto; Kobayashi, Tetsuro; Ohtaka, Manami; Nishimura, Ken; Kurosawa, Hiroshi; Yoshikawa, Takeo; Takahashi, Takuya; Nakanishi, Mahito; Ohyama, Manabu; Hattori, Nobutaka; Akamatsu, Wado; Okano, Hideyuki

    2016-03-01

    Modeling of neurological diseases using induced pluripotent stem cells (iPSCs) derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs). We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinson's disease exhibited several Parkinson's disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases. PMID:26905201

  1. Functional Neurons Generated from T Cell-Derived Induced Pluripotent Stem Cells for Neurological Disease Modeling

    PubMed Central

    Matsumoto, Takuya; Fujimori, Koki; Andoh-Noda, Tomoko; Ando, Takayuki; Kuzumaki, Naoko; Toyoshima, Manabu; Tada, Hirobumi; Imaizumi, Kent; Ishikawa, Mitsuru; Yamaguchi, Ryo; Isoda, Miho; Zhou, Zhi; Sato, Shigeto; Kobayashi, Tetsuro; Ohtaka, Manami; Nishimura, Ken; Kurosawa, Hiroshi; Yoshikawa, Takeo; Takahashi, Takuya; Nakanishi, Mahito; Ohyama, Manabu; Hattori, Nobutaka; Akamatsu, Wado; Okano, Hideyuki

    2016-01-01

    Summary Modeling of neurological diseases using induced pluripotent stem cells (iPSCs) derived from the somatic cells of patients has provided a means of elucidating pathogenic mechanisms and performing drug screening. T cells are an ideal source of patient-specific iPSCs because they can be easily obtained from samples. Recent studies indicated that iPSCs retain an epigenetic memory relating to their cell of origin that restricts their differentiation potential. The classical method of differentiation via embryoid body formation was not suitable for T cell-derived iPSCs (TiPSCs). We developed a neurosphere-based robust differentiation protocol, which enabled TiPSCs to differentiate into functional neurons, despite differences in global gene expression between TiPSCs and adult human dermal fibroblast-derived iPSCs. Furthermore, neurons derived from TiPSCs generated from a juvenile patient with Parkinson's disease exhibited several Parkinson's disease phenotypes. Therefore, we conclude that TiPSCs are a useful tool for modeling neurological diseases. PMID:26905201

  2. Modeling Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived-Cardiomyocytes.

    PubMed

    Maillet, Agnes; Tan, Kim; Chai, Xiaoran; Sadananda, Singh N; Mehta, Ashish; Ooi, Jolene; Hayden, Michael R; Pouladi, Mahmoud A; Ghosh, Sujoy; Shim, Winston; Brunham, Liam R

    2016-01-01

    Doxorubicin is a highly efficacious anti-cancer drug but causes cardiotoxicity in many patients. The mechanisms of doxorubicin-induced cardiotoxicity (DIC) remain incompletely understood. We investigated the characteristics and molecular mechanisms of DIC in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). We found that doxorubicin causes dose-dependent increases in apoptotic and necrotic cell death, reactive oxygen species production, mitochondrial dysfunction and increased intracellular calcium concentration. We characterized genome-wide changes in gene expression caused by doxorubicin using RNA-seq, as well as electrophysiological abnormalities caused by doxorubicin with multi-electrode array technology. Finally, we show that CRISPR-Cas9-mediated disruption of TOP2B, a gene implicated in DIC in mouse studies, significantly reduces the sensitivity of hPSC-CMs to doxorubicin-induced double stranded DNA breaks and cell death. These data establish a human cellular model of DIC that recapitulates many of the cardinal features of this adverse drug reaction and could enable screening for protective agents against DIC as well as assessment of genetic variants involved in doxorubicin response. PMID:27142468

  3. Automated Electrophysiological and Pharmacological Evaluation of Human Pluripotent Stem Cell-Derived Cardiomyocytes.

    PubMed

    Rajamohan, Divya; Kalra, Spandan; Duc Hoang, Minh; George, Vinoj; Staniforth, Andrew; Russell, Hugh; Yang, Xuebin; Denning, Chris

    2016-03-15

    Automated planar patch clamp systems are widely used in drug evaluation studies because of their ability to provide accurate, reliable, and reproducible data in a high-throughput manner. Typically, CHO and HEK tumorigenic cell lines overexpressing single ion channels are used since they can be harvested as high-density, homogenous, single-cell suspensions. While human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) are physiologically more relevant, these cells are fragile, have complex culture requirements, are inherently heterogeneous, and are expensive to produce, which has restricted their use on automated patch clamp (APC) devices. Here, we used high efficiency differentiation protocols to produce cardiomyocytes from six different hPSC lines for analysis on the Patchliner (Nanion Technologies GmbH) APC platform. We developed a two-step cell preparation protocol that yielded cell catch rates and whole-cell breakthroughs of ∼80%, with ∼40% of these cells allowing electrical activity to be recorded. The protocol permitted formation of long-lasting (>15 min), high quality seals (>2 GΩ) in both voltage- and current-clamp modes. This enabled density of sodium, calcium, and potassium currents to be evaluated, along with dose-response curves to their respective channel inhibitors, tetrodotoxin, nifedipine, and E-4031. Thus, we show the feasibility of using the Patchliner platform for automated evaluation of the electrophysiology and pharmacology of hPSC-CMs, which will enable considerable increase in throughput for reliable and efficient drug evaluation. PMID:26906236

  4. Potential of laryngeal muscle regeneration using induced pluripotent stem cell-derived skeletal muscle cells.

    PubMed

    Dirja, Bayu Tirta; Yoshie, Susumu; Ikeda, Masakazu; Imaizumi, Mitsuyoshi; Nakamura, Ryosuke; Otsuki, Koshi; Nomoto, Yukio; Wada, Ikuo; Hazama, Akihiro; Omori, Koichi

    2016-04-01

    Conclusion Induced pluripotent stem (iPS) cells may be a new potential cell source for laryngeal muscle regeneration in the treatment of vocal fold atrophy after recurrent laryngeal nerve paralysis. Objectives Unilateral vocal fold paralysis can lead to degeneration, atrophy, and loss of force of the thyroarytenoid muscle. At present, there are some treatments such as thyroplasty, arytenoid adduction, and vocal fold injection. However, such treatments cannot restore reduced mass of the thyroarytenoid muscle. iPS cells have been recognized as supplying a potential resource for cell transplantation. The aim of this study was to assess the effectiveness of the use of iPS cells for the regeneration of laryngeal muscle through the evaluation of both in vitro and in vivo experiments. Methods Skeletal muscle cells were generated from tdTomato-labeled iPS cells using embryoid body formation. Differentiation into skeletal muscle cells was analyzed by gene expression and immunocytochemistry. The tdTomato-labeled iPS cell-derived skeletal muscle cells were transplanted into the left atrophied thyroarytenoid muscle. To evaluate the engraftment of these cells after transplantation, immunohistochemistry was performed. Results The tdTomato-labeled iPS cells were successfully differentiated into skeletal muscle cells through an in vitro experiment. These cells survived in the atrophied thyroarytenoid muscle after transplantation. PMID:26824385

  5. Process Extension from Embryonic Stem Cell-Derived Motor Neurons through Synthetic Extracellular Matrix Mimics

    NASA Astrophysics Data System (ADS)

    McKinnon, Daniel Devaud

    This thesis focuses on studying the extension of motor axons through synthetic poly(ethylene glycol) PEG hydrogels that have been modified with biochemical functionalities to render them more biologically relevant. Specifically, the research strategy is to encapsulate embryonic stem cell-derived motor neurons (ESMNs) in synthetic PEG hydrogels crosslinked through three different chemistries providing three mechanisms for dynamically tuning material properties. First, a covalently crosslinked, enzymatically degradable hydrogel is developed and exploited to study the biophysical dynamics of axon extension and matrix remodeling. It is demonstrated that dispersed motor neurons require a battery of adhesive peptides and growth factors to maintain viability and extend axons while those in contact with supportive neuroglial cells do not. Additionally, cell-degradable crosslinker peptides and a soft modulus mimicking that of the spinal cord are requirements for axon extension. However, because local degradation of the hydrogel results in a cellular environment significantly different than that of the bulk, enzymatically degradable peptide crosslinkers were replaced with reversible covalent hydrazone bonds to study the effect of hydrogel modulus on axon extension. This material is characterized in detail and used to measure forces involved in axon extension. Finally, a hydrogel with photocleavable linkers incorporated into the network structure is exploited to explore motor axon response to physical channels. This system is used to direct the growth of motor axons towards co-cultured myotubes, resulting in the formation of an in vitro neural circuit.

  6. Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells.

    PubMed

    Rezania, Alireza; Bruin, Jennifer E; Arora, Payal; Rubin, Allison; Batushansky, Irina; Asadi, Ali; O'Dwyer, Shannon; Quiskamp, Nina; Mojibian, Majid; Albrecht, Tobias; Yang, Yu Hsuan Carol; Johnson, James D; Kieffer, Timothy J

    2014-11-01

    Transplantation of pancreatic progenitors or insulin-secreting cells derived from human embryonic stem cells (hESCs) has been proposed as a therapy for diabetes. We describe a seven-stage protocol that efficiently converts hESCs into insulin-producing cells. Stage (S) 7 cells expressed key markers of mature pancreatic beta cells, including MAFA, and displayed glucose-stimulated insulin secretion similar to that of human islets during static incubations in vitro. Additional characterization using single-cell imaging and dynamic glucose stimulation assays revealed similarities but also notable differences between S7 insulin-secreting cells and primary human beta cells. Nevertheless, S7 cells rapidly reversed diabetes in mice within 40 days, roughly four times faster than pancreatic progenitors. Therefore, although S7 cells are not fully equivalent to mature beta cells, their capacity for glucose-responsive insulin secretion and rapid reversal of diabetes in vivo makes them a promising alternative to pancreatic progenitor cells or cadaveric islets for the treatment of diabetes. PMID:25211370

  7. Embryonic stem (ES) cell-derived cardiomyocytes: a good candidate for cell therapy applications.

    PubMed

    Pal, Rajarshi

    2009-03-01

    During the last decade, embryonic stem cells (ESC) have unleashed new avenues in the field of developmental biology and emerged as a potential tool to understand the molecular mechanisms taking place during the process of differentiation from the embryonic stage to adult phenotype. Their uniqueness lies in retaining the capacity of unlimited proliferation and to differentiate into all somatic cells. Together with promising results from rodent models, ESC has raised great hope among for human ESC-based cell replacement therapy. ESC could potentially revolutionize medicine by providing a powerful and renewable cell source capable of replacing or repairing tissues that have been damaged in almost all degenerative diseases such as Parkinson's disease, myocardial infarction (MI) and diabetes. Somatic stem cells are an attractive option to explore for transplantation because they are autologous, but their differentiation potential is very limited. Currently, the major sources of somatic cells used for basic research and clinical trials come from bone marrow. But their widespread acceptability has not been gained because many of the results are confusing and inconsistent. The focus here is on human embryonic stem cells (hESCs), using methods to induce their differentiation to cardiomyocytes in vitro. Their properties in relation to primary human cardiomyocytes and their ability to integrate into host myocardium have been investigated into how they can enhance cardiac function. However, important aspects of stem cell biology and the transplantation process remain unresolved. In summary, this review updates the recent progress of ES cell research in cell therapy, discusses the problems in the practical utility of ESC, and evaluates how far this adjunctive experimental approach can be successful. PMID:19121644

  8. Modeling Cardiovascular Diseases with Patient-Specific Human Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Burridge, Paul W.; Diecke, Sebastian; Matsa, Elena; Sharma, Arun; Wu, Haodi; Wu, Joseph C.

    2016-01-01

    The generation of cardiomyocytes from human induced pluripotent stem cells (hiPSCs) provides a source of cells that accurately recapitulate the human cardiac pathophysiology. The application of these cells allows for modeling of cardiovascular diseases, providing a novel understanding of human disease mechanisms and assessment of therapies. Here, we describe a stepwise protocol developed in our laboratory for the generation of hiPSCs from patients with a specific disease phenotype, long-term hiPSC culture and cryopreservation, differentiation of hiPSCs to cardiomyocytes, and assessment of disease phenotypes. Our protocol combines a number of innovative tools that include a codon-optimized mini intronic plasmid (CoMiP), chemically defined culture conditions to achieve high efficiencies of reprogramming and differentiation, and calcium imaging for assessment of cardiomyocyte phenotypes. Thus, this protocol provides a complete guide to use a patient cohort on a testable cardiomyocyte platform for pharmacological drug assessment. PMID:25690476

  9. PGC-1α and reactive oxygen species regulate human embryonic stem cell-derived cardiomyocyte function.

    PubMed

    Birket, Matthew J; Casini, Simona; Kosmidis, Georgios; Elliott, David A; Gerencser, Akos A; Baartscheer, Antonius; Schumacher, Cees; Mastroberardino, Pier G; Elefanty, Andrew G; Stanley, Ed G; Mummery, Christine L

    2013-01-01

    Diminished mitochondrial function is causally related to some heart diseases. Here, we developed a human disease model based on cardiomyocytes from human embryonic stem cells (hESCs), in which an important pathway of mitochondrial gene expression was inactivated. Repression of PGC-1α, which is normally induced during development of cardiomyocytes, decreased mitochondrial content and activity and decreased the capacity for coping with energetic stress. Yet, concurrently, reactive oxygen species (ROS) levels were lowered, and the amplitude of the action potential and the maximum amplitude of the calcium transient were in fact increased. Importantly, in control cardiomyocytes, lowering ROS levels emulated this beneficial effect of PGC-1α knockdown and similarly increased the calcium transient amplitude. Our results suggest that controlling ROS levels may be of key physiological importance for recapitulating mature cardiomyocyte phenotypes, and the combination of bioassays used in this study may have broad application in the analysis of cardiac physiology pertaining to disease. PMID:24371810

  10. Immaturity of human stem-cell-derived cardiomyocytes in culture: fatal flaw or soluble problem?

    PubMed

    Veerman, Christiaan C; Kosmidis, Georgios; Mummery, Christine L; Casini, Simona; Verkerk, Arie O; Bellin, Milena

    2015-05-01

    Cardiomyocytes from human pluripotent stem cells (hPSC-CMs) are increasingly used to model cardiac disease, test drug efficacy and for safety pharmacology. Nevertheless, a major hurdle to more extensive use is their immaturity and similarity to fetal rather than adult cardiomyocytes. Here, we provide an overview of the strategies currently being used to increase maturation in culture, which include prolongation of time in culture, exposure to electrical stimulation, application of mechanical strain, growth in three-dimensional tissue configuration, addition of non-cardiomyocytes, use of hormones and small molecules, and alteration of the extracellular environment. By comparing the outcomes of these studies, we identify the approaches most likely to improve functional maturation of hPSC-CMs in terms of their electrophysiology and excitation-contraction coupling. PMID:25583389

  11. PGC-1α and Reactive Oxygen Species Regulate Human Embryonic Stem Cell-Derived Cardiomyocyte Function

    PubMed Central

    Birket, Matthew J.; Casini, Simona; Kosmidis, Georgios; Elliott, David A.; Gerencser, Akos A.; Baartscheer, Antonius; Schumacher, Cees; Mastroberardino, Pier G.; Elefanty, Andrew G.; Stanley, Ed G.; Mummery, Christine L.

    2013-01-01

    Summary Diminished mitochondrial function is causally related to some heart diseases. Here, we developed a human disease model based on cardiomyocytes from human embryonic stem cells (hESCs), in which an important pathway of mitochondrial gene expression was inactivated. Repression of PGC-1α, which is normally induced during development of cardiomyocytes, decreased mitochondrial content and activity and decreased the capacity for coping with energetic stress. Yet, concurrently, reactive oxygen species (ROS) levels were lowered, and the amplitude of the action potential and the maximum amplitude of the calcium transient were in fact increased. Importantly, in control cardiomyocytes, lowering ROS levels emulated this beneficial effect of PGC-1α knockdown and similarly increased the calcium transient amplitude. Our results suggest that controlling ROS levels may be of key physiological importance for recapitulating mature cardiomyocyte phenotypes, and the combination of bioassays used in this study may have broad application in the analysis of cardiac physiology pertaining to disease. PMID:24371810

  12. Cardiac Regeneration and Stem Cells.

    PubMed

    Zhang, Yiqiang; Mignone, John; MacLellan, W Robb

    2015-10-01

    After decades of believing the heart loses the ability to regenerate soon after birth, numerous studies are now reporting that the adult heart may indeed be capable of regeneration, although the magnitude of new cardiac myocyte formation varies greatly. While this debate has energized the field of cardiac regeneration and led to a dramatic increase in our understanding of cardiac growth and repair, it has left much confusion in the field as to the prospects of regenerating the heart. Studies applying modern techniques of genetic lineage tracing and carbon-14 dating have begun to establish limits on the amount of endogenous regeneration after cardiac injury, but the underlying cellular mechanisms of this regeneration remained unclear. These same studies have also revealed an astonishing capacity for cardiac repair early in life that is largely lost with adult differentiation and maturation. Regardless, this renewed focus on cardiac regeneration as a therapeutic goal holds great promise as a novel strategy to address the leading cause of death in the developed world. PMID:26269526

  13. Cardiomyocyte MEA data analysis (CardioMDA)--a novel field potential data analysis software for pluripotent stem cell derived cardiomyocytes.

    PubMed

    Pradhapan, Paruthi; Kuusela, Jukka; Viik, Jari; Aalto-Setälä, Katriina; Hyttinen, Jari

    2013-01-01

    Cardiac safety pharmacology requires in-vitro testing of all drug candidates before clinical trials in order to ensure they are screened for cardio-toxic effects which may result in severe arrhythmias. Micro-electrode arrays (MEA) serve as a complement to current in-vitro methods for drug safety testing. However, MEA recordings produce huge volumes of data and manual analysis forms a bottleneck for high-throughput screening. To overcome this issue, we have developed an offline, semi-automatic data analysis software, 'Cardiomyocyte MEA Data Analysis (CardioMDA)', equipped with correlation analysis and ensemble averaging techniques to improve the accuracy, reliability and throughput rate of analysing human pluripotent stem cell derived cardiomyocyte (CM) field potentials. With the program, true field potential and arrhythmogenic complexes can be distinguished from one another. The averaged field potential complexes, analysed using our software to determine the field potential duration, were compared with the analogous values obtained from manual analysis. The reliability of the correlation analysis algorithm, evaluated using various arrhythmogenic and morphology changing signals, revealed a mean sensitivity and specificity of 99.27% and 94.49% respectively, in determining true field potential complexes. The field potential duration of the averaged waveforms corresponded well to the manually analysed data, thus demonstrating the reliability of the software. The software has also the capability to create overlay plots for signals recorded under different drug concentrations in order to visualize and compare the magnitude of response on different ion channels as a result of drug treatment. Our novel field potential analysis platform will facilitate the analysis of CM MEA signals in semi-automated way and provide a reliable means of efficient and swift analysis for cardiomyocyte drug or disease model studies. PMID:24069215

  14. Evaluation of the Cardiotoxicity of Mitragynine and Its Analogues Using Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Wu, Jianjun; Jamil, Mohd Fadzly Amar; Tan, Mei Lan; Adenan, Mohd Ilham; Wong, Philip; Shim, Winston

    2014-01-01

    Introduction Mitragynine is a major bioactive compound of Kratom, which is derived from the leave extracts of Mitragyna speciosa Korth or Mitragyna speciosa (M. speciosa), a medicinal plant from South East Asia used legally in many countries as stimulant with opioid-like effects for the treatment of chronic pain and opioid-withdrawal symptoms. Fatal incidents with Mitragynine have been associated with cardiac arrest. In this study, we determined the cardiotoxicity of Mitragynine and other chemical constituents isolated using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Methods and Results The rapid delayed rectifier potassium current (IKr), L-type Ca2+ current (ICa,L) and action potential duration (APD) were measured by whole cell patch-clamp. The expression of KCNH2 and cytotoxicity was determined by real-time PCR and Caspase activity measurements. After significant IKr suppression by Mitragynine (10 µM) was confirmed in hERG-HEK cells, we systematically examined the effects of Mitragynine and other chemical constituents in hiPSC-CMs. Mitragynine, Paynantheine, Speciogynine and Speciociliatine, dosage-dependently (0.1∼100 µM) suppressed IKr in hiPSC-CMs by 67% ∼84% with IC50 ranged from 0.91 to 2.47 µM. Moreover, Mitragynine (10 µM) significantly prolonged APD at 50 and 90% repolarization (APD50 and APD90) (439.0±11.6 vs. 585.2±45.5 ms and 536.0±22.6 vs. 705.9±46.1 ms, respectively) and induced arrhythmia, without altering the L-type Ca2+ current. Neither the expression,and intracellular distribution of KCNH2/Kv11.1, nor the Caspase 3 activity were significantly affected by Mitragynine. Conclusions Our study indicates that Mitragynine and its analogues may potentiate Torsade de Pointes through inhibition of IKr in human cardiomyocytes. PMID:25535742

  15. Ginsenosides may enhance the functionality of human embryonic stem cell-derived cardiomyocytes in vitro.

    PubMed

    Kim, Yoon Young; Ku, Jun Beom; Liu, Hung Ching; Ku, Seung-Yup; Kim, Seok Hyun; Choi, Young Min

    2014-10-01

    Various chemicals have been reported to induce the differentiation of human embryonic stem cells (hESCs) into cardiomyocytes (CMs), however, their contributions to the functionality of hESC-derived CMs are still limited. In this study, we evaluated the effects of red ginseng extract (RGE), ginsenoside-Rb1 (gRb1, panaxadiol), and ginsenoside-Re (gRe, panaxatriol) on the differentiation of hESCs and the functionality of derived CMs. Undifferentiated hESCs were treated with 0.25 mg/mL RGE, 10 μmol/L gRb1, or 10 μmol/L gRe for 48 hours at the differentiation induction (early stage) or maturation (late stage) period. The expression of mesodermal and cardiac transcription factor genes was upregulated in the ginsenoside-treated groups from early stage. The expression of cardiac sarcomeric genes was significantly upregulated at the late stage. The gRb1- and gRe-treated groups upregulated the expression of potassium voltage-gated channel subfamily E member 1 (KCNE1) and the gRe-treated group showed a longer beating duration compared to the control. Taken together, ginsenosides may enhance the functionality of hESC-derived CMs in vitro. PMID:24615935

  16. Functional Integration of Grafted Neural Stem Cell-Derived Dopaminergic Neurons Monitored by Optogenetics in an In Vitro Parkinson Model

    PubMed Central

    Tønnesen, Jan; Parish, Clare L.; Sørensen, Andreas T.; Andersson, Angelica; Lundberg, Cecilia; Deisseroth, Karl; Arenas, Ernest; Lindvall, Olle; Kokaia, Merab

    2011-01-01

    Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinson's disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D2 autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD. PMID:21394212

  17. Ion channelopathies in human induced pluripotent stem cell derived cardiomyocytes: a dynamic clamp study with virtual IK1

    PubMed Central

    Meijer van Putten, Rosalie M. E.; Mengarelli, Isabella; Guan, Kaomei; Zegers, Jan G.; van Ginneken, Antoni C. G.; Verkerk, Arie O.; Wilders, Ronald

    2015-01-01

    Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) are widely used in studying basic mechanisms of cardiac arrhythmias that are caused by ion channelopathies. Unfortunately, the action potential profile of hiPSC-CMs—and consequently the profile of individual membrane currents active during that action potential—differs substantially from that of native human cardiomyocytes, largely due to almost negligible expression of the inward rectifier potassium current (IK1). In the present study, we attempted to “normalize” the action potential profile of our hiPSC-CMs by inserting a voltage dependent in silico IK1 into our hiPSC-CMs, using the dynamic clamp configuration of the patch clamp technique. Recordings were made from single hiPSC-CMs, using the perforated patch clamp technique at physiological temperature. We assessed three different models of IK1, with different degrees of inward rectification, and systematically varied the magnitude of the inserted IK1. Also, we modified the inserted IK1 in order to assess the effects of loss- and gain-of-function mutations in the KCNJ2 gene, which encodes the Kir2.1 protein that is primarily responsible for the IK1 channel in human ventricle. For our experiments, we selected spontaneously beating hiPSC-CMs, with negligible IK1 as demonstrated in separate voltage clamp experiments, which were paced at 1 Hz. Upon addition of in silico IK1 with a peak outward density of 4–6 pA/pF, these hiPSC-CMs showed a ventricular-like action potential morphology with a stable resting membrane potential near −80 mV and a maximum upstroke velocity >150 V/s (n = 9). Proarrhythmic action potential changes were observed upon injection of both loss-of-function and gain-of-function IK1, as associated with Andersen–Tawil syndrome type 1 and short QT syndrome type 3, respectively (n = 6). We conclude that injection of in silico IK1 makes the hiPSC-CM a more reliable model for investigating mechanisms underlying cardiac

  18. Analysis of Signaling Endosome Composition and Dynamics Using SILAC in Embryonic Stem Cell-Derived Neurons*

    PubMed Central

    Debaisieux, Solène; Encheva, Vesela; Chakravarty, Probir; Snijders, Ambrosius P.; Schiavo, Giampietro

    2016-01-01

    Neurons require efficient transport mechanisms such as fast axonal transport to ensure neuronal homeostasis and survival. Neurotrophins and their receptors are conveyed via fast axonal retrograde transport of signaling endosomes to the soma, where they elicit transcriptional responses. Despite the essential roles of signaling endosomes in neuronal differentiation and survival, little is known about their molecular identity, dynamics, and regulation. Gaining a better mechanistic understanding of these organelles and their kinetics is crucial, given the growing evidence linking vesicular trafficking deficits to neurodegeneration. Here, we exploited an affinity purification strategy using the binding fragment of tetanus neurotoxin (HCT) conjugated to monocrystalline iron oxide nanoparticles (MIONs), which in motor neurons, is transported in the same carriers as neurotrophins and their receptors. To quantitatively assess the molecular composition of HCT-containing signaling endosomes, we have developed a protocol for triple Stable Isotope Labeling with Amino acids in Cell culture (SILAC) in embryonic stem cell-derived motor neurons. After HCT internalization, retrograde carriers were magnetically isolated at different time points and subjected to mass-spectrometry and Gene Ontology analyses. This purification strategy is highly specific, as confirmed by the presence of essential regulators of fast axonal transport in the make-up of these organelles. Our results indicate that signaling endosomes undergo a rapid maturation with the acquisition of late endosome markers following a specific time-dependent kinetics. Strikingly, signaling endosomes are specifically enriched in proteins known to be involved in neurodegenerative diseases and neuroinfection. Moreover, we highlighted the presence of novel components, whose precise temporal recruitment on signaling endosomes might be essential for proper sorting and/or transport of these organelles. This study provides the first

  19. Functionally Enhanced Human Stem Cell Derived Hepatocytes in Galactosylated Cellulosic Sponges for Hepatotoxicity Testing.

    PubMed

    Tasnim, Farah; Toh, Yi-Chin; Qu, Yinghua; Li, Huan; Phan, Derek; Narmada, Balakrishnan C; Ananthanarayanan, Abhishek; Mittal, Nikhil; Meng, Ryan Q; Yu, Hanry

    2016-06-01

    Pluripotent stem cell derived hepatocyte-like cells (hPSC-HLCs) are an attractive alternative to primary human hepatocytes (PHHs) used in applications ranging from therapeutics to drug safety testing studies. It would be critical to improve and maintain mature hepatocyte functions of the hPSC-HLCs, especially for long-term studies. If 3D culture systems were to be used for such purposes, it would be important that the system can support formation and maintenance of optimal-sized spheroids for long periods of time, and can also be directly deployed in liver drug testing assays. We report the use of 3-dimensional (3D) cellulosic scaffold system for the culture of hPSC-HLCs. The scaffold has a macroporous network which helps to control the formation and maintenance of the spheroids for weeks. Our results show that culturing hPSC-HLCs in 3D cellulosic scaffolds increases functionality, as demonstrated by improved urea production and hepatic marker expression. In addition, hPSC-HLCs in the scaffolds exhibit a more mature phenotype, as shown by enhanced cytochrome P450 activity and induction. This enables the system to show a higher sensitivity to hepatotoxicants and a higher degree of similarity to PHHs when compared to conventional 2D systems. These results suggest that 3D cellulosic scaffolds are ideal for the long-term cultures needed to mature hPSC-HLCs. The mature hPSC-HLCs with improved cellular function can be continually maintained in the scaffolds and directly used for hepatotoxicity assays, making this system highly attractive for drug testing applications. PMID:27157693

  20. Plasmid-based transient human stromal cell-derived factor-1 gene transfer improves cardiac function in chronic heart failure

    PubMed Central

    Sundararaman, S; Miller, T J; Pastore, J M; Kiedrowski, M; Aras, R; Penn, M S

    2011-01-01

    We previously demonstrated that transient stromal cell-derived factor-1 alpha (SDF-1) improved cardiac function when delivered via cell therapy in ischemic cardiomyopathy at a time remote from acute myocardial infarction (MI) rats. We hypothesized that non-viral gene transfer of naked plasmid DNA-expressing hSDF-1 could similarly improve cardiac function. To optimize plasmid delivery, we tested SDF-1 and luciferase plasmids driven by the cytomegalovirus (CMV) promoter with (pCMVe) or without (pCMV) translational enhancers or α myosin heavy chain (pMHC) promoter in a rodent model of heart failure. In vivo expression of pCMVe was 10-fold greater than pCMV and pMHC expression and continued over 30 days. We directly injected rat hearts with SDF-1 plasmid 1 month after MI and assessed heart function. At 4 weeks after plasmid injection, we observed a 35.97 and 32.65% decline in fractional shortening (FS) in control (saline) animals and pMHC-hSDF1 animals, respectively, which was sustained to 8 weeks. In contrast, we observed a significant 24.97% increase in animals injected with the pCMVe-hSDF1 vector. Immunohistochemistry of cardiac tissue revealed a significant increase in vessel density in the hSDF-1-treated animals compared with control animals. Increasing SDF-1 expression promoted angiogenesis and improved cardiac function in rats with ischemic heart failure along with evidence of scar remodeling with a trend toward decreased myocardial fibrosis. These data demonstrate that stand-alone non-viral hSDF-1 gene transfer is a strategy for improving cardiac function in ischemic cardiomyopathy. PMID:21472007

  1. Human Embryonic Stem Cells and Cardiac Repair

    PubMed Central

    Zhu, Wei-Zhong; Hauch, Kip; Xu, Chunhui; Laflamme, Michael A.

    2008-01-01

    The muscle lost after a myocardial infarction is replaced with non-contractile scar tissue, often initiating heart failure. Whole-organ cardiac transplantation is the only currently available clinical means of replacing the lost muscle, but this option is limited by the inadequate supply of donor hearts. Thus, cell-based cardiac repair has attracted considerable interest as an alternative means of ameliorating cardiac injury. Because of their tremendous capacity for expansion and unquestioned cardiac potential, pluripotent human embryonic stem cells (hESCs) represent an attractive candidate cell source for obtaining cardiomyocytes and other useful mesenchymal cell types for such therapies. hESC-derived cardiomyocytes (hESC-CMs) exhibit a committed cardiac phenotype and robust proliferative capacity, and recent testing in rodent infarct models indicates that they can partially remuscularize injured hearts and improve contractile function. Although the latter successes give good reason for optimism, considerable challenges remain to the successful application of hESCs to cardiac repair, including the need for preparations of high cardiac purity, improved methods of delivery, and approaches to overcome immune rejection and other causes of graft cell death. This review will describe the phenotype of hESC-CMs and preclinical experience with these cells and will consider strategies to overcoming the aforementioned challenges. PMID:18657407

  2. Potent Paracrine Effects of human induced Pluripotent Stem Cell-derived Mesenchymal Stem Cells Attenuate Doxorubicin-induced Cardiomyopathy

    PubMed Central

    Zhang, Yuelin; Liang, Xiaoting; Liao, Songyan; Wang, Weixin; Wang, Junwen; Li, Xiang; Ding, Yue; Liang, Yingmin; Gao, Fei; Yang, Mo; Fu, Qingling; Xu, Aimin; Chai, Yuet-Hung; He, Jia; Tse, Hung-Fat; Lian, Qizhou

    2015-01-01

    Transplantation of bone marrow mesenchymal stem cells (BM-MSCs) can protect cardiomyocytes against anthracycline-induced cardiomyopathy (AIC) through paracrine effects. Nonetheless the paracrine effects of human induced pluripotent stem cell-derived MSCs (iPSC-MSCs) on AIC are poorly understood. In vitro studies reveal that doxorubicin (Dox)-induced reactive oxidative stress (ROS) generation and cell apoptosis in neonatal rat cardiomyocytes (NRCMs) are significantly reduced when treated with conditioned medium harvested from BM-MSCs (BM-MSCs-CdM) or iPSC-MSCs (iPSC-MSCs-CdM). Compared with BM-MSCs-CdM, NRCMs treated with iPSC-MSCs-CdM exhibit significantly less ROS and cell apoptosis in a dose-dependent manner. Transplantation of BM-MSCs-CdM or iPSC-MSCs-CdM into mice with AIC remarkably attenuated left ventricular (LV) dysfunction and dilatation. Compared with BM-MSCs-CdM, iPSC-MSCs-CdM treatment showed better alleviation of heart failure, less cardiomyocyte apoptosis and fibrosis. Analysis of common and distinct cytokines revealed that macrophage migration inhibitory factor (MIF) and growth differentiation factor-15 (GDF-15) were uniquely overpresented in iPSC-MSC-CdM. Immunodepletion of MIF and GDF-15 in iPSC-MSCs-CdM dramatically decreased cardioprotection. Injection of GDF-15/MIF cytokines could partially reverse Dox-induced heart dysfunction. We suggest that the potent paracrine effects of iPSC-MSCs provide novel “cell-free” therapeutic cardioprotection against AIC, and that MIF and GDF-15 in iPSC-MSCs-CdM are critical for these enhanced cardioprotective effects. PMID:26057572

  3. Liensinine- and Neferine-Induced Cardiotoxicity in Primary Neonatal Rat Cardiomyocytes and Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Yu, Yangyang; Sun, Shennan; Wang, Shifeng; Zhang, Qiao; Li, Ming; Lan, Feng; Li, Shiyou; Liu, Chunsheng

    2016-01-01

    Due to drug-induced potential congestive heart failure and irreversible dilated cardiomyopathies, preclinical evaluation of cardiac dysfunction is important to assess the safety of traditional or novel treatments. The embryos of Nelumbo nucifera Gaertner seeds are a homology of traditional Chinese medicine and food. In this study, we applied the real time cellular analysis (RTCA) Cardio system, which can real-time monitor the contractility of cardiomyocytes (CMs), to evaluate drug safety in rat neonatal CMs and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). This study showed detailed biomechanical CM contractility in vitro, and provided insights into the cardiac dysfunctions associated with liensinine and neferine treatment. These effects exhibited dose and time-dependent recovery. Neferine showed stronger blocking effect in rat neonatal CMs than liensinine. In addition, the effects of liensinine and neferine were further evaluated on hiPS-CMs. Our study also indicated that both liensinine and neferine can cause disruption of calcium homeostasis. For the first time, we demonstrated the potential cardiac side effects of liensinine or neferine. While the same inhibition was observed on hiPS-CMs, more importantly, this study introduced an efficient and effective approach to evaluate the cardiotoxicity of the existing and novel drug candidates. PMID:26840304

  4. Liensinine- and Neferine-Induced Cardiotoxicity in Primary Neonatal Rat Cardiomyocytes and Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

    PubMed

    Yu, Yangyang; Sun, Shennan; Wang, Shifeng; Zhang, Qiao; Li, Ming; Lan, Feng; Li, Shiyou; Liu, Chunsheng

    2016-01-01

    Due to drug-induced potential congestive heart failure and irreversible dilated cardiomyopathies, preclinical evaluation of cardiac dysfunction is important to assess the safety of traditional or novel treatments. The embryos of Nelumbo nucifera Gaertner seeds are a homology of traditional Chinese medicine and food. In this study, we applied the real time cellular analysis (RTCA) Cardio system, which can real-time monitor the contractility of cardiomyocytes (CMs), to evaluate drug safety in rat neonatal CMs and human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). This study showed detailed biomechanical CM contractility in vitro, and provided insights into the cardiac dysfunctions associated with liensinine and neferine treatment. These effects exhibited dose and time-dependent recovery. Neferine showed stronger blocking effect in rat neonatal CMs than liensinine. In addition, the effects of liensinine and neferine were further evaluated on hiPS-CMs. Our study also indicated that both liensinine and neferine can cause disruption of calcium homeostasis. For the first time, we demonstrated the potential cardiac side effects of liensinine or neferine. While the same inhibition was observed on hiPS-CMs, more importantly, this study introduced an efficient and effective approach to evaluate the cardiotoxicity of the existing and novel drug candidates. PMID:26840304

  5. Stem cell-derived vasculature: A potent and multidimensional technology for basic research, disease modeling, and tissue engineering.

    PubMed

    Lowenthal, Justin; Gerecht, Sharon

    2016-05-01

    Proper blood vessel networks are necessary for constructing and re-constructing tissues, promoting wound healing, and delivering metabolic necessities throughout the body. Conversely, an understanding of vascular dysfunction has provided insight into the pathogenesis and progression of diseases both common and rare. Recent advances in stem cell-based regenerative medicine - including advances in stem cell technologies and related progress in bioscaffold design and complex tissue engineering - have allowed rapid advances in the field of vascular biology, leading in turn to more advanced modeling of vascular pathophysiology and improved engineering of vascularized tissue constructs. In this review we examine recent advances in the field of stem cell-derived vasculature, providing an overview of stem cell technologies as a source for vascular cell types and then focusing on their use in three primary areas: studies of vascular development and angiogenesis, improved disease modeling, and the engineering of vascularized constructs for tissue-level modeling and cell-based therapies. PMID:26427871

  6. Induction of Cardiomyogenesis in Human Embryonic Stem Cells by Human Embryonic Stem Cell-Derived Definitive Endoderm

    PubMed Central

    Van Orman, Jordan R.; Si-Tayeb, Karim; Duncan, Stephen A.

    2012-01-01

    We previously reported that chick anterolateral endoderm (AL endoderm) induces cardiomyogenesis in mouse embryoid bodies. However, the requirement to micro-dissect AL endoderm from gastrulation-stage embryos precludes its use to identify novel cardiomyogenic factors, or to scale up cardiomyocyte numbers for therapeutic experiments. To circumvent this problem we have addressed whether human definitive endoderm (hDE) cells, which can be efficiently generated in large numbers from human embryonic stem cells (hESCs), can mimic the ability of AL endoderm to induce cardiac myogenesis. Results demonstrate that both hDE cells and medium conditioned by them induce cardiac myogenesis in pluripotent hESCs, as indicated by rhythmic beating and immunohistochemical/quantitative polymerase chain reaction monitoring of marker gene expression. The cardiomyogenic effect of hDE is enhanced when pluripotent hESCs are preinduced to the mes-endoderm state. Because this approach is tractable and scalable, it may facilitate identification of novel hDE-secreted factors for inclusion in defined cardiomyogenic cocktails. PMID:21627569

  7. Maximum Diastolic Potential of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Depends Critically on IKr

    PubMed Central

    Goodrow, Robert J.; Wu, Yuesheng; Cordeiro, Jonathan M.; Nesterenko, Vladislav V.; Barajas-Martínez, Héctor; Hu, Dan; Urrutia, Janire; Desai, Mayurika; Treat, Jacqueline A.; Sachinidis, Agapios; Antzelevitch, Charles

    2012-01-01

    Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) hold promise for therapeutic applications. To serve these functions, the hiPSC-CM must recapitulate the electrophysiologic properties of native adult cardiomyocytes. This study examines the electrophysiologic characteristics of hiPSC-CM between 11 and 121 days of maturity. Embryoid bodies (EBs) were generated from hiPS cell line reprogrammed with Oct4, Nanog, Lin28 and Sox2. Sharp microelectrodes were used to record action potentials (AP) from spontaneously beating clusters (BC) micro-dissected from the EBs (n = 103; 37°C) and to examine the response to 5 µM E-4031 (n = 21) or BaCl2 (n = 22). Patch-clamp techniques were used to record IKr and IK1 from cells enzymatically dissociated from BC (n = 49; 36°C). Spontaneous cycle length (CL) and AP characteristics varied widely among the 103 preparations. E-4031 (5 µM; n = 21) increased Bazett-corrected AP duration from 291.8±81.2 to 426.4±120.2 msec (p<0.001) and generated early afterdepolarizations in 8/21 preparations. In 13/21 BC, E-4031 rapidly depolarized the clusters leading to inexcitability. BaCl2, at concentrations that selectively block IK1 (50–100 µM), failed to depolarize the majority of clusters (13/22). Patch-clamp experiments revealed very low or negligible IK1 in 53% (20/38) of the cells studied, but presence of IKr in all (11/11). Consistent with the electrophysiological data, RT-PCR and immunohistochemistry studies showed relatively poor mRNA and protein expression of IK1 in the majority of cells, but robust expression of IKr. In contrast to recently reported studies, our data point to major deficiencies of hiPSC-CM, with remarkable diversity of electrophysiologic phenotypes as well as pharmacologic responsiveness among beating clusters and cells up to 121 days post-differentiation (dpd). The vast majority have a maximum diastolic potential that depends critically on IKr due to the absence of IK1. Thus

  8. Endothelial cells derived from embryonic stem cells respond to cues from topographical surface patterns

    PubMed Central

    2013-01-01

    The generation of micro- and nano-topography similar to those found in the extra cellular matrix of three-dimensional tissues is one technique used to recapitulate the cell-tissue physiology found in the native tissues. Despite the fact that ample studies have been conducted on the physiological significance of endothelial cells alignment parallel to shear stress, as this is the normal physiologic arrangement for healthy arterial EC, very few studies have examined the use of topographical signals to initiate endothelial cell alignment. Here, we have examined the ability for our mouse embryonic stem cell-derived endothelial cells (ESC-EC) to align on various microchip topographical systems. Briefly, we generated metal molds with ‘wrinkled’ topography using 1) 15 nm and 2) 30 nm of gold coating on the pre-strained polystryene (PS) sheets. After thermal-induced shrinkage of the PS sheets, polydimethylsiloxane (PDMS) microchips were then generated from the wrinkled molds. Using similar Shrink™-based technology, 3) larger selectively crazed acetone-etched lines in the PS sheets, and 4) fully crazed acetone-treated PS sheets of stochastic topographical morphology were also generated. The 15 nm and 30 nm gold coating generated ‘wrinkles’ of uniaxial anisotropic channels at nano-scaled widths while the crazing generated micron-sized channels. The ESC-EC were able to respond and align on the 320 nm, 510 nm, and the acetone-etched 10.5 μm channels, but not on the fully ‘crazed’ topographies. Moreover, the ESC-EC aligned most robustly on the wrinkles, and preferentially to ridge edges on the 10.5 μm-sized channels. The ability to robustly align EC on topographical surfaces enables a variety of controlled physiological studies of EC-EC and EC-ECM contact guidance, as well as having potential applications for the rapid endothelialization of stents and vascular grafts. PMID:23819656

  9. Endothelial cells derived from embryonic stem cells respond to cues from topographical surface patterns.

    PubMed

    Hatano, Rachel; Mercurio, Kevin; Luna, Jesus Isaac; Glaser, Drew E; Leppert, Valerie J; McCloskey, Kara E

    2013-01-01

    The generation of micro- and nano-topography similar to those found in the extra cellular matrix of three-dimensional tissues is one technique used to recapitulate the cell-tissue physiology found in the native tissues. Despite the fact that ample studies have been conducted on the physiological significance of endothelial cells alignment parallel to shear stress, as this is the normal physiologic arrangement for healthy arterial EC, very few studies have examined the use of topographical signals to initiate endothelial cell alignment. Here, we have examined the ability for our mouse embryonic stem cell-derived endothelial cells (ESC-EC) to align on various microchip topographical systems. Briefly, we generated metal molds with 'wrinkled' topography using 1) 15 nm and 2) 30 nm of gold coating on the pre-strained polystryene (PS) sheets. After thermal-induced shrinkage of the PS sheets, polydimethylsiloxane (PDMS) microchips were then generated from the wrinkled molds. Using similar Shrink™-based technology, 3) larger selectively crazed acetone-etched lines in the PS sheets, and 4) fully crazed acetone-treated PS sheets of stochastic topographical morphology were also generated. The 15 nm and 30 nm gold coating generated 'wrinkles' of uniaxial anisotropic channels at nano-scaled widths while the crazing generated micron-sized channels. The ESC-EC were able to respond and align on the 320 nm, 510 nm, and the acetone-etched 10.5 μm channels, but not on the fully 'crazed' topographies. Moreover, the ESC-EC aligned most robustly on the wrinkles, and preferentially to ridge edges on the 10.5 μm-sized channels. The ability to robustly align EC on topographical surfaces enables a variety of controlled physiological studies of EC-EC and EC-ECM contact guidance, as well as having potential applications for the rapid endothelialization of stents and vascular grafts. PMID:23819656

  10. Innovation in basic science: stem cells and their role in the treatment of paediatric cardiac failure--opportunities and challenges.

    PubMed

    Kaushal, Sunjay; Jacobs, Jeffrey Phillip; Gossett, Jeffrey G; Steele, Ann; Steele, Peter; Davis, Craig R; Pahl, Elfriede; Vijayan, Kalpana; Asante-Korang, Alfred; Boucek, Robert J; Backer, Carl L; Wold, Loren E

    2009-11-01

    Heart failure is a leading cause of death worldwide. Current therapies only delay progression of the cardiac disease or replace the diseased heart with cardiac transplantation. Stem cells represent a recently discovered novel approach to the treatment of cardiac failure that may facilitate the replacement of diseased cardiac tissue and subsequently lead to improved cardiac function and cardiac regeneration. A stem cell is defined as a cell with the properties of being clonogenic, self-renewing, and multipotent. In response to intercellular signalling or environmental stimuli, stem cells differentiate into cells derived from any of the three primary germ layers: ectoderm, endoderm, and mesoderm, a powerful advantage for regenerative therapies. Meanwhile, a cardiac progenitor cell is a multipotent cell that can differentiate into cells of any of the cardiac lineages, including endothelial cells and cardiomyocytes. Stem cells can be classified into three categories: (1) adult stem cells, (2) embryonic stem cells, and (3) induced pluripotential cells. Adult stem cells have been identified in numerous organs and tissues in adults, including bone-marrow, skeletal muscle, adipose tissue, and, as was recently discovered, the heart. Embryonic stem cells are derived from the inner cell mass of the blastocyst stage of the developing embryo. Finally through transcriptional reprogramming, somatic cells, such as fibroblasts, can be converted into induced pluripotential cells that resemble embryonic stem cells. Four classes of stem cells that may lead to cardiac regeneration are: (1) Embryonic stem cells, (2) Bone Marrow derived stem cells, (3) Skeletal myoblasts, and (4) Cardiac stem cells and cardiac progenitor cells. Embryonic stem cells are problematic because of several reasons: (1) the formation of teratomas, (2) potential immunologic cellular rejection, (3) low efficiency of their differentiation into cardiomyocytes, typically 1% in culture, and (4) ethical and political

  11. Loss of Tie2 receptor compromises embryonic stem cell-derived endothelial but not hematopoietic cell survival.

    PubMed

    Hamaguchi, Isao; Morisada, Tohru; Azuma, Masaki; Murakami, Kyoko; Kuramitsu, Madoka; Mizukami, Takuo; Ohbo, Kazuyuki; Yamaguchi, Kazunari; Oike, Yuichi; Dumont, Daniel J; Suda, Toshio

    2006-02-01

    Tie2 is a receptor-type tyrosine kinase expressed on hematopoietic stem cells and endothelial cells. We used cultured embryonic stem (ES) cells to determine the function of Tie2 during early vascular development and hematopoiesis. Upon differentiation, the ES cell-derived Tie2+ Flk1+ fraction was enriched for hematopoietic and endothelial progenitor cells. To investigate lymphatic differentiation, we used a monoclonal antibody against LYVE-1 and found that LYVE-1+ cells derived from Tie2+ Flk1+ cells possessed various characteristics of lymphatic endothelial cells. To determine whether Tie2 played a role in this process, we analyzed differentiation of Tie2-/- ES cells. Although the initial numbers of LYVE-1+ and PECAM-1+ cells derived from Tie2-/- cells did not vary significantly, the number of both decreased dramatically upon extended culturing. Such decreases were rescued by treatment with a caspase inhibitor, suggesting that reductions were due to apoptosis as a consequence of a lack of Tie2 signaling. Interestingly, Tie2-/- ES cells did not show measurable defects in development of the hematopoietic system, suggesting that Tie2 is not essential for hematopoietic cell development. PMID:16219799

  12. Stem cell-derived vascular endothelial cells and their potential application in regenerative medicine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Although a 'vascular stem cell' population has not been identified or generated, vascular endothelial and mural cells (smooth muscle cells and pericytes) can be derived from currently known pluripotent stem cell sources, including human embryonic stem cells and induced pluripotent stem cells. We rev...

  13. Inositol-1,4,5-trisphosphate-mediated spontaneous activity in mouse embryonic stem cell-derived cardiomyocytes.

    PubMed

    Kapur, Nidhi; Banach, Kathrin

    2007-06-15

    Embryonic stem cell-derived cardiomyocytes (ESdCs) have been proposed as a source for cardiac cell-replacement therapy. The aim of this study was to determine the Ca2+-handling mechanisms that determine the frequency and duration of spontaneous Ca2+ transients in single ESdCs. With laser scanning confocal microscopy using the Ca2+-sensitive dye Fluo-4/AM, we determined that spontaneous Ca2+ transients in ESdCs at the onset of beating (day 9) depend on Ca2+ entry across the plasma membrane (50%) whereas Ca2+-induced Ca2+ release is the major contributor to Ca2+ transients in ESdCs after 16 days (72%). Likewise, Ca2+ extrusion in 9-day-old ESdCs depends on Na+-Ca2+ exchange (50.0+/-8%) whereas Ca2+ reuptake by the sarco(endo)plasmic Ca2+ ATPase (72+/-5%) dominates in further differentiated cells. Spontaneous Ca2+ transients were suppressed by the inositol-1,4,5-trisphosphate (IP3) receptor (IP3R) blocker 2-aminoethoxydiphenyl borate (2-APB) and the phospholipase C blocker U73122 but continued in the presence of caffeine. Stimulation of IP3 production by phenylephrine or endothelin-1 had a positive chronotropic effect that could be reversed by U73122 and 2-APB. The presence of Ca2+-free solution and block of L-type Ca2+ channels by nifedipine also resulted in a cessation of spontaneous activity. Overall, IP3R-mediated Ca2+ release in ESdCs is translated into a depolarization of the plasma membrane and a whole-cell Ca2+ transient is subsequently induced by voltage-dependent Ca2+ influx. Although ryanodine receptor-mediated Ca2+ release amplifies the IP3R-induced trigger for the Ca2+ transients and modulates its frequencies, it is not a prerequisite for spontaneous activity. The results of this study offer important insight into the role of IP3R-mediated Ca2+ release for pacemaker activity in differentiating cardiomyocytes. PMID:17379641

  14. Marked hyperglycemia attenuates anesthetic preconditioning in human induced pluripotent stem cell-derived cardiomyocytes

    PubMed Central

    Canfield, Scott G.; Sepac, Ana; Sedlic, Filip; Muravyeva, Maria Y.; Bai, Xiaowen; Bosnjak, Zeljko J.

    2012-01-01

    Introduction Anesthetic preconditioning protects cardiomyocytes from oxidative stress-induced injury, but it is ineffective in patients with diabetes mellitus. To address the role of hyperglycemia in the inability of diabetic individuals to be preconditioned, we used human cardiomyocytes differentiated from induced pluripotent stem cells generated from patients with or without type 2 diabetes mellitus (DM-iPSC- and N-iPSC-CMs, respectively) to investigate the efficacy of preconditioning in varying glucose conditions (5, 11, and 25 mM). Methods Induced pluripotent stem cells were induced to generate cardiomyocytes by directed differentiation. For subsequent studies, cardiomyocytes were identified by genetic labeling with enhanced green fluorescent protein driven by a cardiac-specific promoter. Cell viability was analyzed by lactate dehydrogenase assay. Confocal microscopy was utilized to measure opening of the mitochondrial permeability transition pore and the mitochondrial adenosine-5′-triphosphate-sensitive potassium channels. Results Isoflurane (0.5 mM) preconditioning protected N-iPSC- and DM-iPSC-CMs from oxidative stress-induced lactate dehydrogenase release and mitochondrial permeability transition pore opening in 5 mM and 11 mM glucose. Isoflurane triggered mitochondrial adenosine-5′-triphosphate-sensitive potassium channel opening in N-iPSC-CMs in 5 mM and 11 mM glucose and in DM-iPSC-CMs in 5 mM glucose. 25 mM glucose disrupts anesthetic preconditioning-mediated protection in DM-iPSC- and N-iPSC-CMs. Conclusions The opening of mitochondrial adenosine-5′-triphosphate-sensitive potassium channels are disrupted in DM-iPSC-CMs in 11 mM and 25 mM glucose and in N-iPSC-CMs in 25 mM glucose. Cardiomyocytes derived from healthy donors and patients with a specific disease, such as diabetes in this study, open possibilities in studying genotype and phenotype related pathologies in a human relevant model. PMID:22820846

  15. Isolation of contractile cardiomyocytes from human pluripotent stem-cell-derived cardiomyogenic cultures using a human NCX1-EGFP reporter.

    PubMed

    Ovchinnikov, Dmitry A; Hidalgo, Alejandro; Yang, Seung-Kwon; Zhang, Xinli; Hudson, James; Mazzone, Stuart B; Chen, Chen; Cooper-White, Justin J; Wolvetang, Ernst J

    2015-01-01

    The prospective isolation of defined contractile human pluripotent stem cell (hPSC)-derived cardiomyocytes is advantageous for regenerative medicine and drug screening applications. Currently, enrichment of cardiomyocyte populations from such cultures can be achieved by combinations of cell surface markers or the labor-intensive genetic modification of cardiac developmental genes, such as NKX2.5 or MYH6, with fluorescent reporters. To create a facile, portable method for the isolation of contractile cardiomyocytes from cardiomyogenic hPSC cultures, we employed a highly conserved cardiac enhancer sequence in the SLC8A1 (NCX1) gene to generate a lentivirally deliverable, antibiotic-selectable NCX1cp-EGFP reporter. We show that human embryonic stem cells (and induced pluripotent stem cells) transduced with the NCX1cp-EGFP reporter cassette exhibit enhanced green fluorescent protein (EGFP) expression in cardiac progenitors from 5 days into the directed cardiac hPSC differentiation protocol, with all reporter-positive cells transitioning to spontaneously contracting foci 3 days later. In subsequent stages of cardiomyocyte maturation, NCX1cp-EGFP expression was exclusively limited to contractile cells expressing high levels of cardiac troponin T (CTNT), MLC2a/v, and α-actinin proteins, and was not present in CD90/THY1(+) cardiac stromal cells or CD31/PECAM(+) endothelial cells. Flow-assisted cytometrically sorted EGFP(+) fractions of differentiated cultures were highly enriched in both early (NKX2.5 and TBX5) and late (CTNT/TNNI2, MYH6, MYH7, NPPA, and MYL2) cardiomyocyte markers, with a significant proportion of cells displaying a ventricular-like action potential pattern in patch-clamp recordings. We conclude that the use of the cardiac-specific promoter of the human SLC8A1(NCX1) gene is an effective strategy to isolate contractile cardiac cells and their progenitors from hPSC-derived cardiomyogenic cultures. PMID:25075536

  16. Isolation and Assessment of Mesenchymal Stem Cells Derived From Bone Marrow: Histologic and Histomorphometric Study in a Canine Periodontal Defect.

    PubMed

    Paknejad, Mojgan; Eslaminejad, Mohamadreza Baghaban; Ghaedi, Baharak; Rokn, Amir-Reza; Khorsand, Afshin; Etemad-Moghadam, Shahroo; Alaeddini, Mojgan; Dehghan, Mohammad Mehdi; Moslemi, Neda; Nowzari, Hessam

    2015-06-01

    The aim of the present study was to investigate an isolation procedure to culture mesenchymal stem cells derived from bone marrow and evaluate their potential in periodontal regeneration. Potential stem cells from bone marrow, aspirated from the iliac crest of nine mongrel canines 1 to 2 years of age, were cultivated. After the examination of surface epitopes of the isolated cells, the total RNA from osteogenic, adipogenic, and chondrogenic cell cultures were analyzed by reverse transcription polymerase chain reaction (RT-PCR) to confirm stem cell gene expressions. 2 × 10(7) mL of the stem cells were loaded on 0.2 mL of anorganic bovine bone mineral (ABBM) granules. In each animal, bilateral acute/chronic intrabony periodontal defects were created surgically and by placement of ligatures around the cervical aspect of the teeth. At week 5, after flap debridement, the bilateral defects were randomly assigned to 2 treatment groups: the control group received ABBM, and the test group received BMSCs-loaded ABBM. Eight weeks after transplantation, regenerative parameters were analyzed histologically and histometrically. The RNA expressions confirmed the cultivation of mesenchymal stem cell. More new cementum and periodontal ligament (PDL) were measured in the test group (cementum: 3.33 ± 0.94 vs 2.03 ± 1.30, P = 0.027; PDL: 2.69 ± 0.73 vs 1.53 ± 1.21, P = 0.026). New bone formation was similar in both groups (2.70 ± 0.86 vs 1.99 ± 1.31; P = 0.193). Mesenchymal stem cells derived from bone marrow should be considered a promising technique for use in patients with periodontal attachment loss and merits further investigations. PMID:24383495

  17. Tumor tropism of intravenously injected human-induced pluripotent stem cell-derived neural stem cells and their gene therapy application in a metastatic breast cancer model.

    PubMed

    Yang, Jing; Lam, Dang Hoang; Goh, Sally Sallee; Lee, Esther Xingwei; Zhao, Ying; Tay, Felix Chang; Chen, Can; Du, Shouhui; Balasundaram, Ghayathri; Shahbazi, Mohammad; Tham, Chee Kian; Ng, Wai Hoe; Toh, Han Chong; Wang, Shu

    2012-05-01

    Human pluripotent stem cells can serve as an accessible and reliable source for the generation of functional human cells for medical therapies. In this study, we used a conventional lentiviral transduction method to derive human-induced pluripotent stem (iPS) cells from primary human fibroblasts and then generated neural stem cells (NSCs) from the iPS cells. Using a dual-color whole-body imaging technology, we demonstrated that after tail vein injection, these human NSCs displayed a robust migratory capacity outside the central nervous system in both immunodeficient and immunocompetent mice and homed in on established orthotopic 4T1 mouse mammary tumors. To investigate whether the iPS cell-derived NSCs can be used as a cellular delivery vehicle for cancer gene therapy, the cells were transduced with a baculoviral vector containing the herpes simplex virus thymidine kinase suicide gene and injected through tail vein into 4T1 tumor-bearing mice. The transduced NSCs were effective in inhibiting the growth of the orthotopic 4T1 breast tumor and the metastatic spread of the cancer cells in the presence of ganciclovir, leading to prolonged survival of the tumor-bearing mice. The use of iPS cell-derived NSCs for cancer gene therapy bypasses the sensitive ethical issue surrounding the use of cells derived from human fetal tissues or human embryonic stem cells. This approach may also help to overcome problems associated with allogeneic transplantation of other types of human NSCs. PMID:22311724

  18. Beta-adrenoceptor subtype dependence of chronotropy in mouse embryonic stem cell-derived cardiomyocytes.

    PubMed

    Ali, N N; Xu, X; Brito-Martins, M; Poole-Wilson, P A; Harding, S E; Fuller, S J

    2004-11-01

    Cardiomyocytes derived from embryonic stem cells (ESCM) have potential both as an experimental model for investigating cardiac physiology and as a source for tissue repair. For both reasons it is important to characterise the responses of these cells, and one of the key modulators of contraction is the beta-adrenergic system. We therefore undertook a detailed study of the response of the spontaneous beating rate of ESCM to beta-adrenoceptor (betaAR) stimulation. Embryoid bodies (EBs) were generated from murine ES line E14Tg2a by the hanging drop method, followed by plating. Spontaneously beating areas were seen starting from 9-14 days after differentiation: the experiments described here were performed on EBs between developmental day 19 and 48. Beating cell layers were seeded with charcoal to allow tracking of movement by a video-edge detection system. Experiments were performed in physiological medium containing 1 mM Ca2+ at 37 degrees C. Isoprenaline (Iso) increased beating rate with an EC50 value of 52 nM. Iso (0.3 microM) increased basal rate from 67 +/- 7 beats per minute (bpm) to 138 +/- 18 bpm, P < 0.001, n = 22. At earlier developmental time points the response to Iso was not maintained through 5 min exposure; this spontaneous desensitisation only being observed before day 36. A repeat application of Iso after a wash period of 20 min produced reproducible effects on beating rate. Subtype dependence of the betaAR response was determined by comparing an initial response with a second in the presence of selective beta1- or beta2AR antagonists. In the presence of the specific beta1AR-blocker CGP 20712A (300 nM) the increase in rate with Iso was reduced from 207 +/- 42% of basal to 128 +/- 13%, P < 0.01. With the beta2AR-blocker ICI 118,551 (50 nM) there was no significant change in Iso response. Exposure to the muscarinic agonist, carbachol (10 microM), inhibited the increase in frequency mediated by isoprenaline, but had mixed stimulatory and inhibitory

  19. Mesenchymal Stem Cells Derived from Dental Tissues vs. Those from Other Sources

    PubMed Central

    Huang, G.T.-J.; Gronthos, S.; Shi, S.

    2009-01-01

    To date, 5 different human dental stem/progenitor cells have been isolated and characterized: dental pulp stem cells (DPSCs), stem cells from exfoliated deciduous teeth (SHED), periodontal ligament stem cells (PDLSCs), stem cells from apical papilla (SCAP), and dental follicle progenitor cells (DFPCs). These post-natal populations have mesenchymal-stem-cell-like (MSC) qualities, including the capacity for self-renewal and multilineage differentiation potential. MSCs derived from bone marrow (BMMSCs) are capable of giving rise to various lineages of cells, such as osteogenic, chondrogenic, adipogenic, myogenic, and neurogenic cells. The dental-tissue-derived stem cells are isolated from specialized tissue with potent capacities to differentiate into odontogenic cells. However, they also have the ability to give rise to other cell lineages similar to, but different in potency from, that of BMMSCs. This article will review the isolation and characterization of the properties of different dental MSC-like populations in comparison with those of other MSCs, such as BMMSCs. Important issues in stem cell biology, such as stem cell niche, homing, and immunoregulation, will also be discussed. PMID:19767575

  20. Selection of Phage Display Peptides Targeting Human Pluripotent Stem Cell-Derived Progenitor Cell Lines.

    PubMed

    Bignone, Paola A; Krupa, Rachel A; West, Michael D; Larocca, David

    2016-01-01

    The ability of human pluripotent stem cells (hPS) to both self-renew and differentiate into virtually any cell type makes them a promising source of cells for cell-based regenerative therapies. However, stem cell identity, purity, and scalability remain formidable challenges that need to be overcome for translation of pluripotent stem cell research into clinical applications. Directed differentiation from hPS cells is inefficient and residual contamination with pluripotent cells that have the potential to form tumors remains problematic. The derivation of scalable (self-renewing) embryonic progenitor stem cell lines offers a solution because they are well defined and clonally pure. Clonally pure progenitor stem cell lines also provide a means for identifying cell surface targeting reagents that are useful for identification, tracking, and repeated derivation of the corresponding progenitor stem cell types from additional hPS cell sources. Such stem cell targeting reagents can then be applied to the manufacture of genetically diverse banks of human embryonic progenitor cell lines for drug screening, disease modeling, and cell therapy. Here we present methods to identify human embryonic progenitor stem cell targeting peptides by selection of phage display libraries on clonal embryonic progenitor cell lines and demonstrate their use for targeting quantum dots (Qdots) for stem cell labeling. PMID:25410289

  1. Monosynaptic Tracing using Modified Rabies Virus Reveals Early and Extensive Circuit Integration of Human Embryonic Stem Cell-Derived Neurons.

    PubMed

    Grealish, Shane; Heuer, Andreas; Cardoso, Tiago; Kirkeby, Agnete; Jönsson, Marie; Johansson, Jenny; Björklund, Anders; Jakobsson, Johan; Parmar, Malin

    2015-06-01

    Human embryonic stem cell (hESC)-derived dopamine neurons are currently moving toward clinical use for Parkinson's disease (PD). However, the timing and extent at which stem cell-derived neurons functionally integrate into existing host neural circuitry after transplantation remain largely unknown. In this study, we use modified rabies virus to trace afferent and efferent connectivity of transplanted hESC-derived neurons in a rat model of PD and report that grafted human neurons integrate into the host neural circuitry in an unexpectedly rapid and extensive manner. The pattern of connectivity resembled that of local endogenous neurons, while ectopic connections were not detected. Revealing circuit integration of human dopamine neurons substantiates their potential use in clinical trials. Additionally, our data present rabies-based tracing as a valuable and widely applicable tool for analyzing graft connectivity that can easily be adapted to analyze connectivity of a variety of different neuronal sources and subtypes in different disease models. PMID:26004633

  2. Identification of nephrotoxic compounds with embryonic stem-cell-derived human renal proximal tubular-like cells.

    PubMed

    Li, Yao; Kandasamy, Karthikeyan; Chuah, Jacqueline Kai Chin; Lam, Yue Ning; Toh, Wei Seong; Oo, Zay Yar; Zink, Daniele

    2014-07-01

    The kidney is a major target for drug-induced toxicity, and the renal proximal tubule is frequently affected. Nephrotoxicity is typically detected only late during drug development, and the nephrotoxic potential of newly approved drugs is often underestimated. A central problem is the lack of preclinical models with high predictivity. Validated in vitro models for the prediction of nephrotoxicity are not available. Major problems are related to the identification of appropriate cell models and end points. As drug-induced kidney injury is associated with inflammatory reactions, we explored the expression of inflammatory markers as end point for renal in vitro models. In parallel, we developed a new cell model. Here, we combined these approaches and developed an in vitro model with embryonic stem-cell-derived human renal proximal tubular-like cells that uses the expression of interleukin (IL)-6 and IL-8 as end points. The predictivity of the model was evaluated with 41 well-characterized compounds. The results revealed that the model predicts proximal tubular toxicity in humans with high accuracy. In contrast, the predictivity was low when well-established standard in vitro assays were used. Together, the results show that high predictivity can be obtained with in vitro models employing pluripotent stem cell-derived human renal proximal tubular-like cells. PMID:24495215

  3. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    NASA Astrophysics Data System (ADS)

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-06-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis.

  4. Optimization of surface-immobilized extracellular matrices for the proliferation of neural progenitor cells derived from induced pluripotent stem cells.

    PubMed

    Komura, Takashi; Kato, Koichi; Konagaya, Shuhei; Nakaji-Hirabayashi, Tadashi; Iwata, Hiroo

    2015-11-01

    Neural progenitor cells derived from induced pluripotent stem cells have been considered as a potential source for cell-transplantation therapy of central nervous disorders. However, efficient methods to expand neural progenitor cells are further required for their clinical applications. In this study, a protein array was fabricated with nine extracellular matrices and used to screen substrates suitable for the expansion of neural progenitor cells derived from mouse induced pluripotent stem cells. The results showed that neural progenitor cells efficiently proliferated on substrates with immobilized laminin-1, laminin-5, or Matrigel. Based on this result, further attempts were made to develop clinically compliant substrates with immobilized polypeptides that mimic laminin-1, one of the most effective extracellular matrices as identified in the array-based screening. We used here recombinant DNA technology to prepare polypeptide containing the globular domain 3 of laminin-1 and immobilized it onto glass-based substrates. Our results showed that neural progenitor cells selectively proliferated on substrate with the immobilized polypeptide while maintaining their differentiated state. PMID:25943789

  5. High-Throughput Single-Cell Derived Sphere Formation for Cancer Stem-Like Cell Identification and Analysis

    PubMed Central

    Chen, Yu-Chih; Ingram, Patrick N.; Fouladdel, Shamileh; McDermott, Sean P.; Azizi, Ebrahim; Wicha, Max S.; Yoon, Euisik

    2016-01-01

    Considerable evidence suggests that many malignancies are driven by a cellular compartment that displays stem cell properties. Cancer stem-like cells (CSCs) can be identified by expression of cell surface markers or enzymatic activity, but these methods are limited by phenotypic heterogeneity and plasticity of CSCs. An alternative phenotypic methodology based on in-vitro sphere formation has been developed, but it is typically labor-intensive and low-throughput. In this work, we present a 1,024-microchamber microfluidic platform for single-cell derived sphere formation. Utilizing a hydrodynamic capturing scheme, more than 70% of the microchambers capture only one cell, allowing for monitoring of sphere formation from heterogeneous cancer cell populations for identification of CSCs. Single-cell derived spheres can be retrieved and dissociated for single-cell analysis using a custom 96-gene panel to probe heterogeneity within the clonal CSC spheres. This microfluidic platform provides reliable and high-throughput sphere formation for CSC identification and downstream clonal analysis. PMID:27292795

  6. Isolation and Characterization of Pluripotent Human Spermatogonial Stem Cell-Derived Cells

    PubMed Central

    Kossack, Nina; Meneses, Juanito; Shefi, Shai; Nguyen, Ha Nam; Chavez, Shawn; Nicholas, Cory; Gromoll, Joerg; Turek, Paul J; Reijo-Pera, Renee A

    2009-01-01

    Several reports have documented the derivation of pluripotent cells (multipotent germline stem cells) from spermatogonial stem cells obtained from the adult mouse testis. These spermatogonia-derived stem cells express embryonic stem cell markers and differentiate to the three primary germ layers, as well as the germline. Data indicate that derivation may involve reprogramming of endogenous spermatogonia in culture. Here, we report the derivation of human multipotent germline stem cells (hMGSCs) from a testis biopsy. The cells express distinct markers of pluripotency, form embryoid bodies that contain derivatives of all three germ layers, maintain a normal XY karyotype, are hypomethylated at the H19 locus, and express high levels of telomerase. Teratoma assays indicate the presence of human cells 8 weeks post-transplantation but limited teratoma formation. Thus, these data suggest the potential to derive pluripotent cells from human testis biopsies but indicate a need for novel strategies to optimize hMGSC culture conditions and reprogramming. PMID:18927477

  7. Plasticity of human menstrual blood stem cells derived from the endometrium.

    PubMed

    Lin, Jian; Xiang, Dennis; Zhang, Jin-long; Allickson, Julie; Xiang, Charlie

    2011-05-01

    Stem cells can be obtained from women's menstrual blood derived from the endometrium. The cells display stem cell markers such as Oct-4, SSEA-4, Nanog, and c-kit (CD117), and have the potent ability to differentiate into various cell types, including the heart, nerve, bone, cartilage, and fat. There has been no evidence of teratoma, ectopic formation, or any immune response after transplantation into an animal model. These cells quickly regenerate after menstruation and secrete many growth factors to display recurrent angiogenesis. The plasticity and safety of the acquired cells have been demonstrated in many studies. Menstrual blood-derived stem cells (MenSCs) provide an alternative source of adult stem cells for research and application in regenerative medicine. Here we summarize the multipotent properties and the plasticities of MenSCs and other endometrial stem cells from recent studies conducted both in vitro and in vivo. PMID:21528491

  8. Prion Protein and Stage Specific Embryo Antigen 1 as Selection Markers to Enrich the Fraction of Murine Embryonic Stem Cell-Derived Cardiomyocytes

    PubMed Central

    Ikeda, Nobuhito; Nakayama, Yuji; Nakazawa, Natsumi; Yoshida, Akio; Ninomiya, Haruaki; Shirayoshi, Yasuaki

    2016-01-01

    Background The prion protein (PrP) might be useful as a tool to collect cardiac progenitor cells derived from embryonic stem (ES) cells. It is also possible that PrP+ cells include undifferentiated cells with a capacity to develop into tumors. Methods PrP+ cells isolated from embryoid bodies (EB) formed by mouse AB1 ES cells were examined using RT–PCR analysis and clonogeneic cell assay. To assess their potential to differentiate into cardiomyocytes, Nkx2.5GFP/+ (hcgp7) cells, another ES cell line that carries the GFP reporter gene in the Nkx2.5 loci, were used. Results PrP+ cells isolated from EB of day 7 and 14 did not express pluripotency markers, but expressed cardiac cell markers, while PrP+ cells isolated from EB of day 21 expressed pluripotency markers. Cultured PrP+ cells isolated from EB of day 21 expressed pluripotency markers to form colonies, whereas those isolated from EB of day 7 and 14 did not. To exclude proliferating cells from PrP+ cells, stage specific embryo antigen 1 (SSEA1) was employed as a second marker. PrP+/SSEA1– cells did not proliferate and expressed cardiac cell markers, while PrP+/SSEA1+ did proliferate. Conclusion PrP+ cells isolated from EB included undifferentiated cells in day 21. PrP+/SSEA1– cells included cardiomyoctes, suggesting PrP and SSEA1 may be useful as markers to enrich the fraction of cardiomyocytes. PMID:27493483

  9. Focus on Extracellular Vesicles: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles

    PubMed Central

    Zhang, Bin; Yeo, Ronne Wee Yeh; Tan, Kok Hian; Lim, Sai Kiang

    2016-01-01

    The intense research focus on stem and progenitor cells could be attributed to their differentiation potential to generate new cells to replace diseased or lost cells in many highly intractable degenerative diseases, such as Alzheimer disease, multiple sclerosis, and heart diseases. However, experimental and clinical studies have increasingly attributed the therapeutic efficacy of these cells to their secretion. While stem and progenitor cells secreted many therapeutic molecules, none of these molecules singly or in combination could recapitulate the functional effects of stem cell transplantations. Recently, it was reported that extracellular vesicles (EVs) could recapitulate the therapeutic effects of stem cell transplantation. Based on the observations reported thus far, the prevailing hypothesis is that stem cell EVs exert their therapeutic effects by transferring biologically active molecules such as proteins, lipids, mRNA, and microRNA from the stem cells to injured or diseased cells. In this respect, stem cell EVs are similar to EVs from other cell types. They are both primarily vehicles for intercellular communication. Therefore, the differentiating factor is likely due to the composition of their cargo. The cargo of EVs from different cell types are known to include a common set of proteins and also proteins that reflect the cell source of the EVs and the physiological or pathological state of the cell source. Hence, elucidation of the stem cell EV cargo would provide an insight into the multiple physiological or biochemical changes necessary to affect the many reported stem cell-based therapeutic outcomes in a variety of experimental models and clinical trials. PMID:26861305

  10. Focus on Extracellular Vesicles: Therapeutic Potential of Stem Cell-Derived Extracellular Vesicles.

    PubMed

    Zhang, Bin; Yeo, Ronne Wee Yeh; Tan, Kok Hian; Lim, Sai Kiang

    2016-01-01

    The intense research focus on stem and progenitor cells could be attributed to their differentiation potential to generate new cells to replace diseased or lost cells in many highly intractable degenerative diseases, such as Alzheimer disease, multiple sclerosis, and heart diseases. However, experimental and clinical studies have increasingly attributed the therapeutic efficacy of these cells to their secretion. While stem and progenitor cells secreted many therapeutic molecules, none of these molecules singly or in combination could recapitulate the functional effects of stem cell transplantations. Recently, it was reported that extracellular vesicles (EVs) could recapitulate the therapeutic effects of stem cell transplantation. Based on the observations reported thus far, the prevailing hypothesis is that stem cell EVs exert their therapeutic effects by transferring biologically active molecules such as proteins, lipids, mRNA, and microRNA from the stem cells to injured or diseased cells. In this respect, stem cell EVs are similar to EVs from other cell types. They are both primarily vehicles for intercellular communication. Therefore, the differentiating factor is likely due to the composition of their cargo. The cargo of EVs from different cell types are known to include a common set of proteins and also proteins that reflect the cell source of the EVs and the physiological or pathological state of the cell source. Hence, elucidation of the stem cell EV cargo would provide an insight into the multiple physiological or biochemical changes necessary to affect the many reported stem cell-based therapeutic outcomes in a variety of experimental models and clinical trials. PMID:26861305

  11. A pure population of lung alveolar epithelial type II cells derived from human embryonic stem cells.

    PubMed

    Wang, Dachun; Haviland, David L; Burns, Alan R; Zsigmond, Eva; Wetsel, Rick A

    2007-03-13

    Alveolar epithelial type II (ATII) cells are small, cuboidal cells that constitute approximately 60% of the pulmonary alveolar epithelium. These cells are crucial for repair of the injured alveolus by differentiating into alveolar epithelial type I cells. ATII cells derived from human ES (hES) cells are a promising source of cells that could be used therapeutically to treat distal lung diseases. We have developed a reliable transfection and culture procedure, which facilitates, via genetic selection, the differentiation of hES cells into an essentially pure (>99%) population of ATII cells (hES-ATII). Purity, as well as biological features and morphological characteristics of normal ATII cells, was demonstrated for the hES-ATII cells, including lamellar body formation, expression of surfactant proteins A, B, and C, alpha-1-antitrypsin, and the cystic fibrosis transmembrane conductance receptor, as well as the synthesis and secretion of complement proteins C3 and C5. Collectively, these data document the successful generation of a pure population of ATII cells derived from hES cells, providing a practical source of ATII cells to explore in disease models their potential in the regeneration and repair of the injured alveolus and in the therapeutic treatment of genetic diseases affecting the lung. PMID:17360544

  12. Stem cell-derived vasculature: A potent and multidimensional technology for basic research, disease modeling, and tissue engineering

    PubMed Central

    Lowenthal, Justin; Gerecht, Sharon

    2016-01-01

    Proper blood vessel networks are necessary for constructing and re-constructing tissues, promoting wound healing, and delivering metabolic necessities throughout the body. Conversely, an understanding of vascular dysfunction has provided insight into the pathogenesis and progression of diseases both common and rare. Recent advances in stem cell-based regenerative medicine – including advances in stem cell technologies and related progress in bioscaffold design and complex tissue engineering – have allowed rapid advances in the field of vascular biology, leading in turn to more advanced modeling of vascular pathophysiology and improved engineering of vascularized tissue constructs. In this review we examine recent advances in the field of stem cell-derived vasculature, providing an overview of stem cell technologies as a source for vascular cell types and then focusing on their use in three primary areas: studies of vascular development and angiogenesis, improved disease modeling, and the engineering of vascularized constructs for tissue-level modeling and cell-based therapies. PMID:26427871

  13. Isolation and characterization of SSEA3(+) stem cells derived from goat skin fibroblasts.

    PubMed

    Yang, Zhongcai; Liu, Jun; Liu, Hongliang; Qiu, Mingning; Liu, Qingqing; Zheng, Liming; Pang, Meijun; Quan, Fusheng; Zhang, Yong

    2013-06-01

    Novel stem cells expressing stage-specific embryonic antigen 3 (SSEA-3) reside among human dermal fibroblasts and are known as multilineage-differentiating stress-enduring (Muse) cells. They enhance the generation efficiency of induced pluripotent stem cells. However, Muse cells have only been found in humans. We aimed to isolate SSEA3-positive cells from terminally differentiated skin fibroblasts of adult goat and determine their pluripotency. Cell clusters from SSEA3(+) populations possessed stem cell-like morphological features and normal karyotypes, were consistently positive for alkaline phosphatase, and expressed stem cell pluripotency markers. These SSEA3(+) cells remained undifferentiated over eight passages in suspension culture and were able to differentiate into cells of all three germ layers in vitro and in vivo. Our combined findings suggest that a subset of adult stem cells expressing SSEA3 also exist among adult goat skin fibroblasts. We are the first to report that multipotent adult goat cells exist among terminally differentiated goat skin in suspension culture. Our results also provide a promising platform for generation of a transgenic goat, because the undifferentiated state of stem cells was thought to be more efficient as donor cells for somatic cell nuclear transfer. PMID:23668861

  14. Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

    PubMed Central

    Hubbard, Kyle; Beske, Phillip; Lyman, Megan; McNutt, Patrick

    2015-01-01

    Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and γ-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay

  15. Human Stem Cell-Derived Skin Progenitors Produce Alpha 2-HS Glycoprotein (Fetuin): A Revolutionary Cosmetic Ingredient.

    PubMed

    Nistor, Gabriel; Poole, Aleksandra J; Draelos, Zoe; Lupo, Mary; Tzikas, Thomas; Liu, Jerome H; Keirstead, Hans S

    2016-05-01

    These studies were designed to determine the effect of stem cell-derived skin lineage precursor secretions on the intrinsic and extrinsic symptoms of human skin aging.
    Human stem cells cultivated in balanced conditions were differentiated into skin lineage precursors, and shown to secrete large amounts of fetuin as well as multiple growth factors beneficial for human skin development and maintenance. The cell secretions were incorporated in two simple cosmetic formulations (serum and lotion) and investigated in an IRB-approved 12-week human trial that included 25 subjects in each group. Subjects were examined at 2, 4, 8, and 12 weeks by a dermatologist to evaluate safety, trans-epidermal water loss, wrinkles, firmness, radiance, texture, softness, and overall appearance. A sub-group of subjects from each group consented for biopsies for histological analyses.
    Protein analyses in the cell secretions revealed a high concentration of the multifunctional alpha 2-HS glycoprotein (fetuin) along with a multitude of protein factors involved in the development and maintenance of healthy human skin. Clinical investigation demonstrated significant amelioration of the clinical signs of intrinsic and extrinsic skin aging, findings that were confirmed by significant changes in skin morphology, filaggrin, aquaporin 3, and collagen I content.
    Our data strongly support our hypothesis that cosmetic application of stem cell-derived skin lineage precursor secretions containing fetuin and growth factors beneficial for human skin development and maintenance, positively influence intrinsic and extrinsic aging.

    J Drugs Dermatol. 2016;15(5):583-598. PMID:27168267

  16. Bio-Oss®acts on Stem cells derived from Peripheral Blood

    PubMed Central

    Sollazzo, Vincenzo; Palmieri, Annalisa; Scapoli, Luca; Martinelli, Marcella; Girardi, Ambra; Alviano, Francesco; Pellati, Agnese; Perrotti, Vittoria; Carinci, Francesco

    2010-01-01

    Objectives This study aims to study how Bio-Oss® can induce osteoblast differentiation in mesenchymal stem cells, the expression levels of bone related genes and mesenchymal stem cells markers using real time Reverse Transcription-Polymerase Chain Reaction. Methods PB-hMSCs stem preparations were obtained for gradient centrifugation from peripheral blood of healthy anonymous volunteers, using the Acuspin System-Histopaque 1077. The samples were then cultured for 7 days for RNA processing, and the expression was quantified using real time PCR. Results Bio-Oss® caused an induction of osteoblast transcriptional factor like RUNX2 and of bone related genes; SPP1 and FOSL1. In contrast, the expression of ENG was significantly decreased in stem cells treated with Bio-Oss® with respect to untreated cells, indicating the differentiation effect of this biomaterial on stem cells. Conclusion The results obtained can be relevant to enhance the understanding of the molecular mechanism of bone regeneration and can act as a model for comparing other materials with similar clinical effects. PMID:22125694

  17. Pluripotent stem cells derived from mouse primordial germ cells by small molecule compounds.

    PubMed

    Kimura, Tohru; Kaga, Yoshiaki; Sekita, Yoichi; Fujikawa, Keita; Nakatani, Tsunetoshi; Odamoto, Mika; Funaki, Soichiro; Ikawa, Masahito; Abe, Kuniya; Nakano, Toru

    2015-01-01

    Primordial germ cells (PGCs) can give rise to pluripotent stem cells known as embryonic germ cells (EGCs) when cultured with basic fibroblast growth factor (bFGF), stem cell factor (SCF), and leukemia inhibitory factor. Somatic cells can give rise to induced pluripotent stem cells (iPSCs) by introduction of the reprogramming transcription factors Oct4, Sox2, and Klf4. The effects of Sox2 and Klf4 on somatic cell reprogramming can be reproduced using the small molecule compounds, transforming growth factor-β receptor (TGFβR) inhibitor and Kempaullone, respectively. Here we examined the effects of TGFβR inhibitor and Kempaullone on EGC derivation from PGCs. Treatment of PGCs with TGFβR inhibitor and/or Kempaullone generated pluripotent stem cells under standard embryonic stem cell (ESC) culture conditions without bFGF and SCF, which we termed induced EGCs (iEGCs). The derivation efficiency of iEGCs was dependent on the differentiation stage and sex. DNA methylation levels of imprinted genes in iEGCs were reduced, with the exception of the H19 gene. The promoters of genes involved in germline development were generally hypomethylated in PGCs, but three germline genes showed comparable DNA methylation levels among iEGs, ESCs, and iPSCs. These results show that PGCs can be reprogrammed into pluripotent state using small molecule compounds, and that DNA methylation of these germline genes is not maintained in iEGCs. PMID:25186651

  18. Neuronal differentiation of embryonic stem cell derived neuronal progenitors can be regulated by stretchable conducting polymers.

    PubMed

    Srivastava, Nishit; Venugopalan, Vijay; Divya, M S; Rasheed, V A; James, Jackson; Narayan, K S

    2013-09-01

    Electrically conducting polymers are prospective candidates as active substrates for the development of neuroprosthetic devices. The utility of these substrates for promoting differentiation of embryonic stem cells paves viable routes for regenerative medicine. Here, we have tuned the electrical and mechanical cues provided to the embryonic stem cells during differentiation by precisely straining the conducting polymer (CP) coated, elastomeric-substrate. Upon straining the substrates, the neural differentiation pattern occurs in form of aggregates, accompanied by a gradient where substrate interface reveals a higher degree of differentiation. The CP domains align under linear stress along with the formation of local defect patterns leading to disruption of actin cytoskeleton of cells, and can provide a mechano-transductive basis for the observed changes in the differentiation. Our results demonstrate that along with biochemical and mechanical cues, conductivity of the polymer plays a major role in cellular differentiation thereby providing another control feature to modulate the differentiation and proliferation of stem cells. PMID:23544950

  19. Neuronal Differentiation of Embryonic Stem Cell Derived Neuronal Progenitors Can Be Regulated by Stretchable Conducting Polymers

    PubMed Central

    Srivastava, Nishit; Venugopalan, Vijay; Divya, M.S.; Rasheed, V.A.

    2013-01-01

    Electrically conducting polymers are prospective candidates as active substrates for the development of neuroprosthetic devices. The utility of these substrates for promoting differentiation of embryonic stem cells paves viable routes for regenerative medicine. Here, we have tuned the electrical and mechanical cues provided to the embryonic stem cells during differentiation by precisely straining the conducting polymer (CP) coated, elastomeric-substrate. Upon straining the substrates, the neural differentiation pattern occurs in form of aggregates, accompanied by a gradient where substrate interface reveals a higher degree of differentiation. The CP domains align under linear stress along with the formation of local defect patterns leading to disruption of actin cytoskeleton of cells, and can provide a mechano-transductive basis for the observed changes in the differentiation. Our results demonstrate that along with biochemical and mechanical cues, conductivity of the polymer plays a major role in cellular differentiation thereby providing another control feature to modulate the differentiation and proliferation of stem cells. PMID:23544950

  20. Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass.

    PubMed

    Guo, Ge; von Meyenn, Ferdinand; Santos, Fatima; Chen, Yaoyao; Reik, Wolf; Bertone, Paul; Smith, Austin; Nichols, Jennifer

    2016-04-12

    Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals. PMID:26947977

  1. Naive Pluripotent Stem Cells Derived Directly from Isolated Cells of the Human Inner Cell Mass

    PubMed Central

    Guo, Ge; von Meyenn, Ferdinand; Santos, Fatima; Chen, Yaoyao; Reik, Wolf; Bertone, Paul; Smith, Austin; Nichols, Jennifer

    2016-01-01

    Summary Conventional generation of stem cells from human blastocysts produces a developmentally advanced, or primed, stage of pluripotency. In vitro resetting to a more naive phenotype has been reported. However, whether the reset culture conditions of selective kinase inhibition can enable capture of naive epiblast cells directly from the embryo has not been determined. Here, we show that in these specific conditions individual inner cell mass cells grow into colonies that may then be expanded over multiple passages while retaining a diploid karyotype and naive properties. The cells express hallmark naive pluripotency factors and additionally display features of mitochondrial respiration, global gene expression, and genome-wide hypomethylation distinct from primed cells. They transition through primed pluripotency into somatic lineage differentiation. Collectively these attributes suggest classification as human naive embryonic stem cells. Human counterparts of canonical mouse embryonic stem cells would argue for conservation in the phased progression of pluripotency in mammals. PMID:26947977

  2. Instructing an Embryonic Stem Cell-Derived Oocyte Fate: Lessons from Endogenous Oogenesis

    PubMed Central

    Nicholas, Cory R.; Chavez, Shawn L.; Baker, Valerie L.; Reijo Pera, Renee A.

    2009-01-01

    Female reproductive potential is limited in the majority of species due to oocyte depletion. Because functional human oocytes are restricted in number and accessibility, a robust system to differentiate oocytes from stem cells would enable a thorough investigation of the genetic, epigenetic, and environmental factors affecting human oocyte development. Also, the differentiation of functional oocytes from stem cells may permit the success of human somatic cell nuclear transfer for reprogramming studies and for the production of patient-specific embryonic stem cells (ESCs). Thus, ESC-derived oocytes could ultimately help to restore fertility in women. Here, we review endogenous and ESC-derived oocyte development, and we discuss the potential and challenges for differentiating functional oocytes from ESCs. PMID:19366753

  3. Human Pluripotent Stem Cell-Derived Cardiomyocytes as Research and Therapeutic Tools

    PubMed Central

    Pesl, Martin; Lacampagne, Alain; Dvorak, Petr; Rotrekl, Vladimir; Meli, Albano C.

    2014-01-01

    Human pluripotent stem cells (hPSCs), namely, embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), with their ability of indefinite self-renewal and capability to differentiate into cell types derivatives of all three germ layers, represent a powerful research tool in developmental biology, for drug screening, disease modelling, and potentially cell replacement therapy. Efficient differentiation protocols that would result in the cell type of our interest are needed for maximal exploitation of these cells. In the present work, we aim at focusing on the protocols for differentiation of hPSCs into functional cardiomyocytes in vitro as well as achievements in the heart disease modelling and drug testing on the patient-specific iPSC-derived cardiomyocytes (iPSC-CMs). PMID:24800237

  4. Akt Suppression of TGFβ Signaling Contributes to the Maintenance of Vascular Identity in Embryonic Stem Cell-Derived Endothelial Cells

    PubMed Central

    Israely, Edo; Ginsberg, Michael; Nolan, Daniel; Ding, Bi-Sen; James, Daylon; Elemento, Olivier; Rafii, Shahin; Rabbany, Sina Y

    2016-01-01

    The ability to generate and maintain stable in vitro cultures of mouse endothelial cells (EC) has great potential for genetic dissection of the numerous pathologies involving vascular dysfunction as well as therapeutic applications. However, previous efforts at achieving sustained cultures of primary stable murine vascular cells have fallen short, and the cellular requirements for EC maintenance in vitro remain undefined. In this study, we have generated vascular ECs from mouse embryonic stem (ES) cells, and show that active Akt is essential to their survival and propagation as homogeneous monolayers in vitro. These cells harbor the phenotypical, biochemical, and functional characteristics of ECs, and expand throughout long-term cultures, while maintaining their angiogenic capacity. Moreover, Akt-transduced embryonic ECs form functional perfused vessels in vivo that anastomose with host blood vessels. We provide evidence for a novel function of Akt in stabilizing EC identity, whereby the activated form of the protein protects mouse ES cell-derived ECs from TGFβ-mediated transdifferentiation by downregulating SMAD3. These findings identify a role for Akt in regulating the developmental potential of ES cell-derived ECs, and demonstrate that active Akt maintains endothelial identity in embryonic ECs by interfering with active TGFβ-mediated processes that would ordinarily usher these cells to alternate fates. PMID:23963623

  5. Defined MicroRNAs Induce Aspects of Maturation in Mouse and Human Embryonic-Stem-Cell-Derived Cardiomyocytes.

    PubMed

    Lee, Desy S; Chen, Jyh-Hong; Lundy, David J; Liu, Chung-Hung; Hwang, Shiaw-Min; Pabon, Lil; Shieh, Ru-Chi; Chen, Chien-Chang; Wu, Sheng-Nan; Yan, Yu-Ting; Lee, Sho-Tone; Chiang, Po-Min; Chien, Shu; Murry, Charles E; Hsieh, Patrick C H

    2015-09-29

    Pluripotent-cell-derived cardiomyocytes have great potential for use in research and medicine, but limitations in their maturity currently constrain their usefulness. Here, we report a method for improving features of maturation in murine and human embryonic-stem-cell-derived cardiomyocytes (m/hESC-CMs). We found that coculturing m/hESC-CMs with endothelial cells improves their maturity and upregulates several microRNAs. Delivering four of these microRNAs, miR-125b-5p, miR-199a-5p, miR-221, and miR-222 (miR-combo), to m/hESC-CMs resulted in improved sarcomere alignment and calcium handling, a more negative resting membrane potential, and increased expression of cardiomyocyte maturation markers. Although this could not fully phenocopy all adult cardiomyocyte characteristics, these effects persisted for two months following delivery of miR-combo. A luciferase assay demonstrated that all four miRNAs target ErbB4, and siRNA knockdown of ErbB4 partially recapitulated the effects of miR-combo. In summary, a combination of miRNAs induced via endothelial coculture improved ESC-CM maturity, in part through suppression of ErbB4 signaling. PMID:26365191

  6. Pluripotency of adult stem cells derived from human and rat pancreas

    NASA Astrophysics Data System (ADS)

    Kruse, C.; Birth, M.; Rohwedel, J.; Assmuth, K.; Goepel, A.; Wedel, T.

    Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases.

  7. Pluripotent Stem Cells Derived From Mouse and Human White Mature Adipocytes

    PubMed Central

    Abdmaulen, Raushan; Ly, Albert; Cubberly, Mark R.; Shahmirian, Laurine J.; Heydarkhan-Hagvall, Sepideh; Dumesic, Daniel A.; Yao, Yucheng

    2014-01-01

    White mature adipocytes give rise to so-called dedifferentiated fat (DFAT) cells that spontaneously undergo multilineage differentiation. In this study, we defined stem cell characteristics of DFAT cells as they are generated from adipocytes and the relationship between these characteristics and lineage differentiation. Both mouse and human DFAT cells, prepared from adipose tissue and lipoaspirate, respectively, showed evidence of pluripotency, with a maximum 5–7 days after adipocyte isolation. The DFAT cells spontaneously formed clusters in culture, which transiently expressed multiple stem cell markers, including stage-specific embryonic antigens, and Sca-1 (mouse) and CD105 (human), as determined by real-time polymerase chain reaction, fluorescence-activated cell sorting, and immunostaining. As the stem cell markers decreased, markers characteristic of the three germ layers and specific lineage differentiation, such as α-fetoprotein (endoderm, hepatic), Neurofilament-66 (ectoderm, neurogenic), and Troponin I (mesoderm, cardiomyogenic), increased. However, no teratoma formation was detected after injection in immunodeficient mice. A novel modification of the adipocyte isolation aimed at ensuring the initial purity of the adipocytes and avoiding ceiling culture allowed isolation of DFAT cells with pluripotent characteristics. Thus, the adipocyte-derived DFAT cells represent a plastic stem cell population that is highly responsive to changes in culture conditions and may benefit cell-based therapies. PMID:24396033

  8. Labeling pluripotent stem cell-derived neural progenitors with iron oxide particles for magnetic resonance imaging.

    PubMed

    Sart, Sébastien; Bejarano, Fabian Calixto; Yan, Yuanwei; Grant, Samuel C; Li, Yan

    2015-01-01

    Due to the unlimited proliferation capacity and the unique differentiation ability of pluripotent stem cells (PSCs), including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), large numbers of PSC-derived cell products are in demand for applications in drug screening, disease modeling, and especially cell therapy. In stem cell-based therapy, tracking transplanted cells with magnetic resonance imaging (MRI) has emerged as a powerful technique to reveal cell survival and distribution. This chapter illustrated the basic steps of labeling PSC-derived neural progenitors (NPs) with micron-sized particles of iron oxide (MPIO, 0.86 μm) for MRI analysis. The protocol described PSC expansion and differentiation into NPs, and the labeling of the derived cells either after replating on adherent surface or in suspension. The labeled cells can be analyzed using in vitro MRI analysis. The methods presented here can be easily adapted for cell labeling in cell processing facilities under current Good Manufacturing Practices (cGMP). The iron oxide-labeled NPs can be used for cellular monitoring of in vitro cultures and in vivo transplantation. PMID:25304204

  9. Slow-Adhering Stem Cells Derived from Injured Skeletal Muscle Have Improved Regenerative Capacity

    PubMed Central

    Mu, Xiaodong; Xiang, Guosheng; Rathbone, Christopher R.; Pan, Haiying; Bellayr, Ian H.; Walters, Thomas J.; Li, Yong

    2011-01-01

    A wide variety of myogenic cell sources have been used for repair of injured and diseased muscle including muscle stem cells, which can be isolated from skeletal muscle as a group of slow-adhering cells on a collagen-coated surface. The therapeutic use of muscle stem cells for improving muscle regeneration is promising; however, the effect of injury on their characteristics and engraftment potential has yet to be described. In the present study, slow-adhering stem cells (SASCs) from both laceration-injured and control noninjured skeletal muscles in mice were isolated and studied. Migration and proliferation rates, multidifferentiation potentials, and differences in gene expression in both groups of cells were compared in vitro. Results demonstrated that a larger population of SASCs could be isolated from injured muscle than from control noninjured muscle. In addition, SASCs derived from injured muscle demonstrated improved migration, a higher rate of proliferation and multidifferentiation, and increased expression of Notch1, STAT3, Msx1, and MMP2. Moreover, when transplanted into dystrophic muscle in MDX/SCID mice, SASCs from injured muscle generated greater engraftments with a higher capillary density than did SASCs from control noninjured muscle. These data suggest that traumatic injury may modify stem cell characteristics through trophic factors and improve the transplantation potential of SASCs in alleviating skeletal muscle injuries and diseases. PMID:21684246

  10. N-butylidenephthalide attenuates Alzheimer's disease-like cytopathy in Down syndrome induced pluripotent stem cell-derived neurons.

    PubMed

    Chang, Chia-Yu; Chen, Sheng-Mei; Lu, Huai-En; Lai, Syu-Ming; Lai, Ping-Shan; Shen, Po-Wen; Chen, Pei-Ying; Shen, Ching-I; Harn, Horng-Jyh; Lin, Shinn-Zong; Hwang, Shiaw-Min; Su, Hong-Lin

    2015-01-01

    Down syndrome (DS) patients with early-onset dementia share similar neurodegenerative features with Alzheimer's disease (AD). To recapitulate the AD cell model, DS induced pluripotent stem cells (DS-iPSCs), reprogrammed from mesenchymal stem cells in amniotic fluid, were directed toward a neuronal lineage. Neuroepithelial precursor cells with high purity and forebrain characteristics were robustly generated on day 10 (D10) of differentiation. Accumulated amyloid deposits, Tau protein hyperphosphorylation and Tau intracellular redistribution emerged rapidly in DS neurons within 45 days but not in normal embryonic stem cell-derived neurons. N-butylidenephthalide (Bdph), a major phthalide ingredient of Angelica sinensis, was emulsified by pluronic F127 to reduce its cellular toxicity and promote canonical Wnt signaling. Interestingly, we found that F127-Bdph showed significant therapeutic effects in reducing secreted Aβ40 deposits, the total Tau level and the hyperphosphorylated status of Tau in DS neurons. Taken together, DS-iPSC derived neural cells can serve as an ideal cellular model of DS and AD and have potential for high-throughput screening of candidate drugs. We also suggest that Bdph may benefit DS or AD treatment by scavenging Aβ aggregates and neurofibrillary tangles. PMID:25735452

  11. N-butylidenephthalide Attenuates Alzheimer's Disease-Like Cytopathy in Down Syndrome Induced Pluripotent Stem Cell-Derived Neurons

    PubMed Central

    Chang, Chia-Yu; Chen, Sheng-Mei; Lu, Huai-En; Lai, Syu-Ming; Lai, Ping-Shan; Shen, Po-Wen; Chen, Pei-Ying; Shen, Ching-I; Harn, Horng-Jyh; Lin, Shinn-Zong; Hwang, Shiaw-Min; Su, Hong-Lin

    2015-01-01

    Down syndrome (DS) patients with early-onset dementia share similar neurodegenerative features with Alzheimer's disease (AD). To recapitulate the AD cell model, DS induced pluripotent stem cells (DS-iPSCs), reprogrammed from mesenchymal stem cells in amniotic fluid, were directed toward a neuronal lineage. Neuroepithelial precursor cells with high purity and forebrain characteristics were robustly generated on day 10 (D10) of differentiation. Accumulated amyloid deposits, Tau protein hyperphosphorylation and Tau intracellular redistribution emerged rapidly in DS neurons within 45 days but not in normal embryonic stem cell-derived neurons. N-butylidenephthalide (Bdph), a major phthalide ingredient of Angelica sinensis, was emulsified by pluronic F127 to reduce its cellular toxicity and promote canonical Wnt signaling. Interestingly, we found that F127-Bdph showed significant therapeutic effects in reducing secreted Aβ40 deposits, the total Tau level and the hyperphosphorylated status of Tau in DS neurons. Taken together, DS-iPSC derived neural cells can serve as an ideal cellular model of DS and AD and have potential for high-throughput screening of candidate drugs. We also suggest that Bdph may benefit DS or AD treatment by scavenging Aβ aggregates and neurofibrillary tangles. PMID:25735452

  12. Human Embryonic Stem Cell-Derived Neural Precursors Develop Into Neurons and Integrate Into the Host Brain

    PubMed Central

    Guillaume, Daniel J.; Johnson, M. Austin; Li, Xue-Jun; Zhang, Su-Chun

    2009-01-01

    Whether and how in-vitro-produced human neural precursors mature and integrate into the brain are crucial to the utility of human embryonic stem (hES) cells in treating neurological disorders. After transplantation into the ventricles of neonatal immune-deficient mice, hES-cell-derived neural precursors stopped expressing the cell division marker Ki67, except in neurogenic areas, and differentiated into neurons and then glia in a temporal course intrinsic to that of human cells regardless of location. The human cells located in the gray matter became neurons in the olfactory bulb and striatum, whereas those in the white matter produced exclusively glia. Importantly, the grafted human cells formed synapses. Thus, the in-vitro-produced human neural precursors follow their intrinsic temporal program to produce neurons and glia and, in response to environmental signals, generate cells appropriate to their target regions and integrate into the brain. PMID:16941479

  13. Early maturation and distinct tau pathology in induced pluripotent stem cell-derived neurons from patients with MAPT mutations.

    PubMed

    Iovino, Mariangela; Agathou, Sylvia; González-Rueda, Ana; Del Castillo Velasco-Herrera, Martin; Borroni, Barbara; Alberici, Antonella; Lynch, Timothy; O'Dowd, Sean; Geti, Imbisaat; Gaffney, Daniel; Vallier, Ludovic; Paulsen, Ole; Káradóttir, Ragnhildur Thóra; Spillantini, Maria Grazia

    2015-11-01

    Tauopathies, such as Alzheimer's disease, some cases of frontotemporal dementia, corticobasal degeneration and progressive supranuclear palsy, are characterized by aggregates of the microtubule-associated protein tau, which are linked to neuronal death and disease development and can be caused by mutations in the MAPT gene. Six tau isoforms are present in the adult human brain and they differ by the presence of 3(3R) or 4(4R) C-terminal repeats. Only the shortest 3R isoform is present in foetal brain. MAPT mutations found in human disease affect tau binding to microtubules or the 3R:4R isoform ratio by altering exon 10 splicing. We have differentiated neurons from induced pluripotent stem cells derived from fibroblasts of controls and patients with N279K and P301L MAPT mutations. Induced pluripotent stem cell-derived neurons recapitulate developmental tau expression, showing the adult brain tau isoforms after several months in culture. Both N279K and P301L neurons exhibit earlier electrophysiological maturation and altered mitochondrial transport compared to controls. Specifically, the N279K neurons show abnormally premature developmental 4R tau expression, including changes in the 3R:4R isoform ratio and AT100-hyperphosphorylated tau aggregates, while P301L neurons are characterized by contorted processes with varicosity-like structures, some containing both alpha-synuclein and 4R tau. The previously unreported faster maturation of MAPT mutant human neurons, the developmental expression of 4R tau and the morphological alterations may contribute to disease development. PMID:26220942

  14. Unique proliferation response in odontoblastic cells derived from human skeletal muscle stem cells by cytokine-induced matrix metalloproteinase-3

    SciTech Connect

    Ozeki, Nobuaki; Hase, Naoko; Kawai, Rie; Yamaguchi, Hideyuki; Hiyama, Taiki; Kondo, Ayami; Nakata, Kazuhiko; Mogi, Makio

    2015-02-01

    A pro-inflammatory cytokine mixture (CM: interleukin (IL)-1β, tumor necrosis factor-α and interferon-γ) and IL-1β-induced matrix metalloproteinase (MMP)-3 activity have been shown to increase the proliferation of rat dental pulp cells and murine stem cell-derived odontoblast-like cells. This suggests that MMP-3 may regulate wound healing and regeneration in the odontoblast-rich dental pulp. Here, we determined whether these results can be extrapolated to human dental pulp by investigating the effects of CM-induced MMP-3 up-regulation on the proliferation and apoptosis of purified odontoblast-like cells derived from human skeletal muscle stem cells. We used siRNA to specifically reduce MMP-3 expression. We found that CM treatment increased MMP-3 mRNA and protein levels as well as MMP-3 activity. Cell proliferation was also markedly increased, with no changes in apoptosis, upon treatment with CM and following the application of exogenous MMP-3. Endogenous tissue inhibitors of metalloproteinases were constitutively expressed during all experiments and unaffected by MMP-3. Although treatment with MMP-3 siRNA suppressed cell proliferation, it also unexpectedly increased apoptosis. This siRNA-mediated increase in apoptosis could be reversed by exogenous MMP-3. These results demonstrate that cytokine-induced MMP-3 activity regulates cell proliferation and suppresses apoptosis in human odontoblast-like cells. - Highlights: • Pro-inflammatory cytokines induce MMP-3 activity in human odontoblast-like cells. • Increased MMP-3 activity can promote cell proliferation in odontoblasts. • Specific loss of MMP-3 increases apoptosis in odontoblasts. • MMP-3 has potential as a promising new target for pupal repair and regeneration.

  15. Early maturation and distinct tau pathology in induced pluripotent stem cell-derived neurons from patients with MAPT mutations

    PubMed Central

    Iovino, Mariangela; Agathou, Sylvia; González-Rueda, Ana; Del Castillo Velasco-Herrera, Martin; Borroni, Barbara; Alberici, Antonella; Lynch, Timothy; O’Dowd, Sean; Geti, Imbisaat; Gaffney, Daniel; Vallier, Ludovic; Paulsen, Ole; Káradóttir, Ragnhildur Thóra

    2015-01-01

    Tauopathies, such as Alzheimer’s disease, some cases of frontotemporal dementia, corticobasal degeneration and progressive supranuclear palsy, are characterized by aggregates of the microtubule-associated protein tau, which are linked to neuronal death and disease development and can be caused by mutations in the MAPT gene. Six tau isoforms are present in the adult human brain and they differ by the presence of 3(3R) or 4(4R) C-terminal repeats. Only the shortest 3R isoform is present in foetal brain. MAPT mutations found in human disease affect tau binding to microtubules or the 3R:4R isoform ratio by altering exon 10 splicing. We have differentiated neurons from induced pluripotent stem cells derived from fibroblasts of controls and patients with N279K and P301L MAPT mutations. Induced pluripotent stem cell-derived neurons recapitulate developmental tau expression, showing the adult brain tau isoforms after several months in culture. Both N279K and P301L neurons exhibit earlier electrophysiological maturation and altered mitochondrial transport compared to controls. Specifically, the N279K neurons show abnormally premature developmental 4R tau expression, including changes in the 3R:4R isoform ratio and AT100-hyperphosphorylated tau aggregates, while P301L neurons are characterized by contorted processes with varicosity-like structures, some containing both alpha-synuclein and 4R tau. The previously unreported faster maturation of MAPT mutant human neurons, the developmental expression of 4R tau and the morphological alterations may contribute to disease development. PMID:26220942

  16. Mesenchymal Stem Cell-Derived Extracellular Vesicles Promote Angiogenesis: Potencial Clinical Application

    PubMed Central

    Merino-González, Consuelo; Zuñiga, Felipe A.; Escudero, Carlos; Ormazabal, Valeska; Reyes, Camila; Nova-Lamperti, Estefanía; Salomón, Carlos; Aguayo, Claudio

    2016-01-01

    Mesenchymal stem cells (MSCs) are adult multipotent stem cells that are able to differentiate into multiple specialized cell types including osteocytes, adipocytes, and chondrocytes. MSCs exert different functions in the body and have recently been predicted to have a major clinical/therapeutic potential. However, the mechanisms of self-renewal and tissue regeneration are not completely understood. It has been shown that the biological effect depends mainly on its paracrine action. Furthermore, it has been reported that the secretion of soluble factors and the release of extracellular vesicles, such as exosomes, could mediate the cellular communication to induce cell-differentiation/self-renewal. This review provides an overview of MSC-derived exosomes in promoting angiogenicity and of the clinical relevance in a therapeutic approach. PMID:26903875

  17. Efficient Generation of Viral and Integration-Free Human Induced Pluripotent Stem Cell-Derived Oligodendrocytes.

    PubMed

    Espinosa-Jeffrey, Araceli; Blanchi, Bruno; Biancotti, Juan Carlos; Kumar, Shalini; Hirose, Megumi; Mandefro, Berhan; Talavera-Adame, Dodanim; Benvenisty, Nissim; de Vellis, Jean

    2016-01-01

    Here we document three highly reproducible protocols: (1) a culture system for the derivation of human oligodendrocytes (OLs) from human induced pluripotent stem cells (hiPS) and their further maturation-our protocol generates viral- and integration-free OLs that efficiently commit and move forward in the OL lineage, recapitulating all the steps known to occur during in vivo development; (2) a method for the isolation, propagation and maintenance of neural stem cells (NSCs); and (3) a protocol for the production, isolation, and maintenance of OLs from perinatal rodent and human brain-derived NSCs. Our unique culture systems rely on a series of chemically defined media, specifically designed and carefully characterized for each developmental stage of OL as they advance from OL progenitors to mature, myelinating cells. We are confident that these protocols bring our field a step closer to efficient autologous cell replacement therapies and disease modeling. © 2016 by John Wiley & Sons, Inc. PMID:27532816

  18. Immune regulation by mesenchymal stem cells derived from adult spleen and thymus.

    PubMed

    Krampera, Mauro; Sartoris, Silvia; Liotta, Francesco; Pasini, Annalisa; Angeli, Roberta; Cosmi, Lorenzo; Andreini, Angelo; Mosna, Federico; Bonetti, Bruno; Rebellato, Elisabetta; Testi, Maria Grazia; Frosali, Francesca; Pizzolo, Giovanni; Tridente, Giuseppe; Maggi, Enrico; Romagnani, Sergio; Annunziato, Francesco

    2007-10-01

    We show here that human and mouse mesenchymal stem cells (MSCs) can be obtained not only from bone marrow (BM), but also from adult spleen and thymus. In vitro, both human and mouse spleen- and thymus-derived MSCs exhibit immunophenotypic characteristics and differentiation potential completely comparable to BM-MSCs. In addition, they can inhibit immune responses mediated by activated T lymphocytes with efficiency comparable to BM-MSCs. In vivo, mouse MSCs from BM, spleen, and thymus, if injected together with a genetically modified tumor cell vaccine, can equally prevent the onset of an anti-tumor memory immune response, thus leading to tumor growth in normally resistant mice. Our data suggest that not only do spleen and thymus have a stem cell reservoir to build up their stromal architecture, but also contain microenviromental immunoregulatory cells with the same properties of BM-MSCs. PMID:17999601

  19. Smooth Muscle Precursor Cells Derived from Human Pluripotent Stem Cells for Treatment of Stress Urinary Incontinence.

    PubMed

    Wang, Zhe; Wen, Yan; Li, Yan Hui; Wei, Yi; Green, Morgaine; Wani, Prachi; Zhang, Pengbo; Pera, Renee Reijo; Chen, Bertha

    2016-03-15

    There is great interest in using stem cells (SC) to regenerate a deficient urethral sphincter in patients with urinary incontinence. The smooth muscle component of the sphincter is a significant contributor to sphincter function. However, current translational efforts for sphincter muscle restoration focus only on skeletal muscle regeneration because they rely on adult mesenchymal SC as cell source. These adult SC do not yield sufficient smooth muscle cells (SMCs) for transplantation. We may be able to overcome this limitation by using pluripotent stem cell (PSC) to derive SMCs. Hence, we sought to investigate whether smooth muscle precursor cells (pSMCs) derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery + saline injection), bladder SMC (surgery + human bladder SMC injection), and treatment (surgery + pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed induced pluripotent stem cells (iPSCs)-derived pSMC, or viral reprogrammed iPSC-derived pSMC). pSMCs (2 × 10(6) cells/rat) were injected periurethrally 3 weeks postsurgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks postinjection. Both iPSC-derived pSMC treatment groups showed significantly higher LPP compared to the sham saline group, consistent with restoration of urethral sphincter function. While the difference between the H9-derived pSMC treatment and sham saline group was not significant, it did show a trend toward restoration of the LPP to the level of intact controls. Our data indicate that pSMCs derived from human PSCs (hESC and iPSC) can restore sphincter function. PMID:26785911

  20. Label-free Electrophysiological Cytometry for Stem Cell-Derived Cardiomyocyte Clusters

    PubMed Central

    Myers, Frank B.; Abilez, Oscar J.; Zarins, Christopher K.; Lee, Luke P.

    2012-01-01

    Stem cell therapies hold great promise for repairing tissues damaged due to disease or injury. However, a major obstacle facing this field is the difficulty in identifying cells of a desired phenotype from the heterogeneous population that arises during stem cell differentiation. Conventional fluorescence flow cytometry and magnetic cell purification require exogenous labeling of cell surface markers which can interfere with the performance of the cells of interest. Here, we describe a non-genetic, label-free cell cytometry method based on electrophysiological response to stimulus. As many of the cell types relevant for regenerative medicine are electrically-excitable (e.g. cardiomyocytes, neurons, smooth muscle cells), this technology is well-suited for identifying cells from heterogeneous stem cell progeny without the risk and expense associated with molecular labeling or genetic modification. Our label-free cell cytometer is capable of distinguishing clusters of undifferentiated human induced pluripotent stem cells (iPSC) from iPSC-derived cardiomyocyte (iPSC-CM) clusters. The system utilizes a microfluidic device with integrated electrodes for both electrical stimulation and recording of extracellular field potential (FP) signals from suspended cells in flow. The unique electrode configuration provides excellent rejection of field stimulus artifact while enabling sensitive detection of FPs with a noise floor of 2 μVrms. Cells are self-aligned to the recording electrodes via hydrodynamic flow focusing. Based on automated analysis of these extracellular signals, the system distinguishes cardiomyocytes from non-cardiomyocytes. This is an entirely new approach to cell cytometry, in which a cell’s functionality is assessed rather than its expression profile or physical characteristics. PMID:23207961

  1. Smooth Muscle Precursor Cells Derived from Human Pluripotent Stem Cells for Treatment of Stress Urinary Incontinence

    PubMed Central

    Wang, Zhe; Li, Yan Hui; Wei, Yi; Green, Morgaine; Wani, Prachi; Zhang, Pengbo; Pera, Renee Reijo; Chen, Bertha

    2016-01-01

    There is great interest in using stem cells (SC) to regenerate a deficient urethral sphincter in patients with urinary incontinence. The smooth muscle component of the sphincter is a significant contributor to sphincter function. However, current translational efforts for sphincter muscle restoration focus only on skeletal muscle regeneration because they rely on adult mesenchymal SC as cell source. These adult SC do not yield sufficient smooth muscle cells (SMCs) for transplantation. We may be able to overcome this limitation by using pluripotent stem cell (PSC) to derive SMCs. Hence, we sought to investigate whether smooth muscle precursor cells (pSMCs) derived from human PSCs can restore urethral function in an animal model generated by surgical urethrolysis and ovariectomy. Rats were divided into four groups: control (no intervention), sham saline (surgery + saline injection), bladder SMC (surgery + human bladder SMC injection), and treatment (surgery + pSMC injection, which includes human embryonic stem cell (hESC) H9-derived pSMC, episomal reprogrammed induced pluripotent stem cells (iPSCs)-derived pSMC, or viral reprogrammed iPSC-derived pSMC). pSMCs (2 × 106 cells/rat) were injected periurethrally 3 weeks postsurgery. Leak point pressure (LPP) and baseline external urethral sphincter electromyography were measured 5 weeks postinjection. Both iPSC-derived pSMC treatment groups showed significantly higher LPP compared to the sham saline group, consistent with restoration of urethral sphincter function. While the difference between the H9-derived pSMC treatment and sham saline group was not significant, it did show a trend toward restoration of the LPP to the level of intact controls. Our data indicate that pSMCs derived from human PSCs (hESC and iPSC) can restore sphincter function. PMID:26785911

  2. Isolation and characterisation of mesenchymal stem cells derived from human placenta tissue

    PubMed Central

    Vellasamy, Shalini; Sandrasaigaran, Pratheep; Vidyadaran, Sharmili; George, Elizabeth; Ramasamy, Rajesh

    2012-01-01

    AIM: To explore the feasibility of placenta tissue as a reliable and efficient source for generating mesenchymal stem cells (MSC). METHODS: MSC were generated from human placenta tissue by enzymatic digestion and mechanical dissociation. The placenta MSC (PLC-MSC) were characterized for expression of cell surface markers, embryonic stem cell (ECS) gene expression and their differentiation ability into adipocytes and osteocytes. The immunosuppressive properties of PLC-MSC on resting and phytohemagglutinin (PHA) stimulated allogenic T cells were assessed by means of cell proliferation via incorporation of tritium thymidine (3H-TdR). RESULTS: The generated PLC-MSC appeared as spindle-shaped cells, expressed common MSC surface markers and ESC transcriptional factors. They also differentiated into adipogenic and osteogenic lineages when induced. However, continuous cultivation up to passage 15 caused changes in morphological appearance and cellular senescence, although the stem cell nature of their protein expression was unchanged. In terms of their immunosuppressive properties, PLC-MSC were unable to stimulate resting T cell proliferation; they inhibited the PHA stimulated T cells in a dose dependent manner through cell to cell contact. In our study, MSC generated from human placenta exhibited similar mesenchymal cell surface markers; MSC-like gene expression pattern and MSC-like differentiation potential were comparable to other sources of MSC. CONCLUSION: We suggest that placenta tissues can serve as an alternative source of MSC for future experimental and clinical studies. PMID:22993662

  3. In Vitro and In Vivo Germ Line Potential of Stem Cells Derived from Newborn Mouse Skin

    PubMed Central

    Dyce, Paul W.; Liu, Jinghe; Tayade, Chandrakant; Kidder, Gerald M.; Betts, Dean H.; Li, Julang

    2011-01-01

    We previously reported that fetal porcine skin-derived stem cells were capable of differentiation into oocyte-like cells (OLCs). Here we report that newborn mice skin-derived stem cells are also capable of differentiating into early OLCs. Using stem cells from mice that are transgenic for Oct4 germline distal enhancer-GFP, germ cells resulting from their differentiation are expected to be GFP+. After differentiation, some GFP+ OLCs reached 40–45 µM and expressed oocyte markers. Flow cytometric analysis revealed that ∼0.3% of the freshly isolated skin cells were GFP+. The GFP-positive cells increased to ∼7% after differentiation, suggesting that the GFP+ cells could be of in vivo origin, but are more likely induced upon being cultured in vitro. To study the in vivo germ cell potential of skin-derived cells, they were aggregated with newborn ovarian cells, and transplanted under the kidney capsule of ovariectomized mice. GFP+ oocytes were identified within a subpopulation of follicles in the resulting growth. Our finding that early oocytes can be differentiated from mice skin-derived cells in defined medium may offer a new in vitro model to study germ cell formation and oogenesis. PMID:21629667

  4. New miRNAs network in human mesenchymal stem cells derived from skin and amniotic fluid.

    PubMed

    Lazzarini, R; Sorgentoni, G; Caffarini, M; Sayeed, M A; Olivieri, F; Di Primio, R; Orciani, M

    2016-09-01

    Mesenchymal stem cells (MSCs), isolated from different adult sources, have great appeal for therapeutic applications due to their simple isolation, extensive expansion potential, and high differentiative potential.In our previous studies we isolated MSCs form amniotic fluid (AF-MSCs) and skin (S-MSCs) and characterized them according to their phenotype, pluripotency, and mRNA/microRNAs (miRNAs) profiling using Card A from Life Technologies.Here, we enlarge the profiling of AF-MCSs and S-MSCs to the more recently discovered miRNAs (Card B by Life Technologies) to identify the miRNAs putative target genes and the relative signaling pathways. Card B, in fact, contains miRNAs whose role and target are not yet elucidated.The expression of the analyzed miRNAs is changing between S-MSCs and AF-MSCs, indicating that these two types of MSCs show differences potentially related to their source. Interestingly, the pathways targeted by the miRNAS deriving from Card B are the same found during the analysis of miRNAs from Card A.This result confirms the key role played by WNT and TGF-β pathways in stem cell fate, underlining as other miRNAs partially ignored up to now deserve to be reconsidered. In addition, this analysis allows including Adherens junction pathways among the mechanisms finely regulated in stem cell behavior. PMID:26684628

  5. Immunological Properties of Corneal Epithelial-Like Cells Derived from Human Embryonic Stem Cells

    PubMed Central

    Wang, Zhenyu; Zhou, Qingjun; Duan, Haoyun; Wang, Yao; Dong, Muchen; Shi, Weiyun

    2016-01-01

    Transplantation of ex vivo expanded corneal limbal stem cells (LSCs) has been the main treatment for limbal stem cell deficiency, although the shortage of donor corneal tissues remains a major concern for its wide application. Due to the development of tissue engineering, embryonic stem cells (ESCs)-derived corneal epithelial-like cells (ESC-CECs) become a new direction for this issue. However, the immunogenicity of ESC-CECs is a critical matter to be solved. In the present study, we explored the immunological properties of ESC-CECs, which were differentiated from ESCs. The results showed that ESC-CECs had a similar character and function with LSCs both in vitro and in vivo. In ESC-CECs, a large number of genes related with immune response were down-regulated. The expressions of MHC-I, MHC-II, and co-stimulatory molecules were low, but the expression of HLA-G was high. The ESC-CECs were less responsible for T cell proliferation and NK cell lysis in vitro, and there was less immune cell infiltration after transplantation in vivo compared with LSCs. Moreover, the immunological properties were not affected by interferon-γ. All these results indicated a low immunogenicity of ESC-CECs, and they can be promising in clinical use. PMID:26977925

  6. Proliferation rate of stem cells derived from human dental pulp and identification of differentially expressed genes.

    PubMed

    Abdullah, Muhammad Fawwaz; Abdullah, Siti Fadilah; Omar, Nor Shamsuria; Mahmood, Zuliani; Fazliah Mohd Noor, Siti Noor; Kannan, Thirumulu Ponnuraj; Mokhtar, Khairani Idah

    2014-05-01

    Stem cells from human exfoliated deciduous teeth (SHED) and dental pulp stem cells (DPSCs) obtained from the dental pulp of human extracted tooth were cultured and characterized to confirm that these were mesenchymal stem cells. The proliferation rate was assessed using AlamarBlue® cell assay. The differentially expressed genes in SHED and DPSCs were identified using the GeneFishing™ technique. The proliferation rate of SHED (P < 0.05) was significantly higher than DPSCs while SHED had a lower multiplication rate and shorter population doubling time (0.01429, 60.57 h) than DPSCs (0.00286, 472.43 h). Two bands were highly expressed in SHED and three bands in DPSCs. Sequencing analysis showed these to be TIMP metallopeptidase inhibitor 1 (TIMP1), and ribosomal protein s8, (RPS8) in SHED and collagen, type I, alpha 1, (COL1A1), follistatin-like 1 (FSTL1), lectin, galactoside-binding, soluble, 1, (LGALS1) in DPSCs. TIMP1 is involved in degradation of the extracellular matrix, cell proliferation and anti-apoptotic function and RPS8 is involved as a rate-limiting factor in translational regulation; COL1A1 is involved in the resistance and elasticity of the tissues; FSTL1 is an autoantigen associated with rheumatoid arthritis; LGALS1 is involved in cell growth, differentiation, adhesion, RNA processing, apoptosis and malignant transformation. This, along with further protein expression analysis, holds promise in tissue engineering and regenerative medicine. PMID:24375868

  7. Ultrastructural maturation of human bone marrow mesenchymal stem cells-derived cardiomyocytes under alternative induction of 5-azacytidine.

    PubMed

    Piryaei, Abbas; Soleimani, Masoud; Heidari, Mohammad Hassan; Saheli, Mona; Rohani, Razieh; Almasieh, Mohammadali

    2015-05-01

    Adult cardiomyocytes lack the ability to proliferate and are unable to repair damaged heart tissue, therefore differentiation of stem cells to cardiomyocytes represents an exceptional opportunity to study cardiomyocytes in vitro and potentially provides a valuable source for replacing damaged tissue. However, characteristic maturity of the in vitro differentiated cardiomyocytes and methods to achieve it are yet to be optimized. In this study, differentiation of human bone marrow-mesenchymal stem cells (hBM-MSCs) into cardiomyocytes is accomplished and the process investigated ultrastructurally. The hBM-MSCs were alternatively treated with 5 μM of 5-azacytidine (5-aza) for 8 weeks resulting in differentiation to cardiomyocytes. Expressions of cardiomyocyte-specific genes [cardiac α-actinin, cardiac β-myosin heavy chain (MHC) and connexin-43] and proteins (cardiac α-actinin, cardiac troponin and connexin-43) were confirmed in a time-dependent manner from the first to the fifth weeks post-induction. Ultrastructural maturation of hBM-MSCs-derived cardiomyocyte (MSCs-CM) corresponded with increase in number and organization of myofilaments in cells over time. Starting from week five, organized myofibrils along with developing sarcomeres were detectable. Later on, MSCs-CM were characterized by the presence of sarcoplasmic reticulum, T-tubules and diads as cardiomyocytes connected to each other by intercalated disc-like structures. Here, we showed the potential of hBM-MSCs as a source for the production of cardiomyocytes and confirmed mature ultrastructural characteristics of these cells using our alternative incubation method. PMID:25573851

  8. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo.

    PubMed

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  9. Porcine Pluripotent Stem Cells Derived from IVF Embryos Contribute to Chimeric Development In Vivo

    PubMed Central

    Xue, Binghua; Li, Yan; He, Yilong; Wei, Renyue; Sun, Ruizhen; Yin, Zhi; Bou, Gerelchimeg; Liu, Zhonghua

    2016-01-01

    Although the pig is considered an important model of human disease and an ideal animal for the preclinical testing of cell transplantation, the utility of this model has been hampered by a lack of genuine porcine embryonic stem cells. Here, we derived a porcine pluripotent stem cell (pPSC) line from day 5.5 blastocysts in a newly developed culture system based on MXV medium and a 5% oxygen atmosphere. The pPSCs had been passaged more than 75 times over two years, and the morphology of the colony was similar to that of human embryonic stem cells. Characterization and assessment showed that the pPSCs were alkaline phosphatase (AKP) positive, possessed normal karyotypes and expressed classic pluripotent markers, including OCT4, SOX2 and NANOG. In vitro differentiation through embryonic body formation and in vivo differentiation via teratoma formation in nude mice demonstrated that the pPSCs could differentiate into cells of the three germ layers. The pPSCs transfected with fuw-DsRed (pPSC-FDs) could be passaged with a stable expression of both DsRed and pluripotent markers. Notably, when pPSC-FDs were used as donor cells for somatic nuclear transfer, 11.52% of the reconstructed embryos developed into blastocysts, which was not significantly different from that of the reconstructed embryos derived from porcine embryonic fibroblasts. When pPSC-FDs were injected into day 4.5 blastocysts, they became involved in the in vitro embryonic development and contributed to the viscera of foetuses at day 50 of pregnancy as well as the developed placenta after the chimeric blastocysts were transferred into recipients. These findings indicated that the pPSCs were porcine pluripotent cells; that this would be a useful cell line for porcine genetic engineering and a valuable cell line for clarifying the molecular mechanism of pluripotency regulation in pigs. PMID:26991423

  10. Interaction of Salmonella Typhimurium with Dendritic Cells Derived from Pluripotent Embryonic Stem Cells

    PubMed Central

    Rossi, Raffaella; Hale, Christine; Goulding, David; Andrews, Robert; Abdellah, Zarah; Fairchild, Paul J.; Dougan, Gordon

    2012-01-01

    Using an in vitro differentiation protocol we isolated cells with the properties of dendritic cells (DCs) from immunologically refractive pluripotent murine embryonic stem cells (ESCs). These ES-derived dendritic cells (ESDCs) expressed cytokines and were able to present antigen to a T cell line. Infection of ESDCs with Salmonella Typhimurium stimulated the expression of immune cell markers and thousands of murine genes, many associated with the immune response. Consequently, this system provides a novel in vitro model, amenable to genetic modification, for monitoring host/pathogen interactions. PMID:23284947