Browsing by Subject "Embryonic Stem Cells"

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    African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast.
    (PloS one, 2018-01) Sheridan, Megan A; Balaraman, Velmurugan; Schust, Danny J; Ezashi, Toshihiko; Roberts, R Michael; Franz, Alexander WE
    Zika virus (ZIKV) drew worldwide attention when a recent epidemic was linked to fetal microcephaly. Here we used human embryonic stem cell derived trophoblasts as a model for primitive placental trophoblast to test the hypothesis that there are differences in how the two genetically distinct ZIKV lineages, African (AF) and Asian (AS), target the human placenta. Upon infection with three AF (ib-H30656, SEN/1984/41525-DAK, and MR-766) and three AS (FSS13025, MexI-44, and PANcdc259249) ZIKV strains, we observed that severe placental cell lysis was only induced after infection with AF strains, while viral replication rates remained similar between both lineages. Differences in cytopathic effects (CPE) were not observed in Vero cells, indicating that the AF strains were not inherently superior at cell lysis. Taken together, we propose that infection with AF strains of ZIKV early in pregnancy would likely result in pregnancy loss, rather than allow further fetal development with accompanying brain damage. Our results also suggest that the long term laboratory-adapted MR-766 strain does not behave aberrantly in cell culture relative to other AF lineage strains.
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    Growth factor erv1-like modulates Drp1 to preserve mitochondrial dynamics and function in mouse embryonic stem cells.
    (Mol Biol Cell, 2010-04-01) Todd, Lance R; Damin, Matthew N; Gomathinayagam, Rohini; Horn, Sarah R; Means, Anthony R; Sankar, Uma
    The relationship of mitochondrial dynamics and function to pluripotency are rather poorly understood aspects of stem cell biology. Here we show that growth factor erv1-like (Gfer) is involved in preserving mouse embryonic stem cell (ESC) mitochondrial morphology and function. Knockdown (KD) of Gfer in ESCs leads to decreased pluripotency marker expression, embryoid body (EB) formation, cell survival, and loss of mitochondrial function. Mitochondria in Gfer-KD ESCs undergo excessive fragmentation and mitophagy, whereas those in ESCs overexpressing Gfer appear elongated. Levels of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) are highly elevated in Gfer-KD ESCs and decreased in Gfer-overexpressing cells. Treatment with a specific inhibitor of Drp1 rescues mitochondrial function and apoptosis, whereas expression of Drp1-dominant negative resulted in the restoration of pluripotency marker expression in Gfer-KD ESCs. Altogether, our data reveal a novel prosurvival role for Gfer in maintaining mitochondrial fission-fusion dynamics in pluripotent ESCs.
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    Heme Oxygenase-1/Carbon Monoxide System and Embryonic Stem Cell Differentiation and Maturation into Cardiomyocytes.
    (Antioxid Redox Signal, 2016-03-01) Suliman, Hagir B; Zobi, Fabio; Piantadosi, Claude A
    AIMS: The differentiation of embryonic stem (ES) cells into energetically efficient cardiomyocytes contributes to functional cardiac repair and is envisioned to ameliorate progressive degenerative cardiac diseases. Advanced cell maturation strategies are therefore needed to create abundant mature cardiomyocytes. In this study, we tested whether the redox-sensitive heme oxygenase-1/carbon monoxide (HO-1/CO) system, operating through mitochondrial biogenesis, acts as a mechanism for ES cell differentiation and cardiomyocyte maturation. RESULTS: Manipulation of HO-1/CO to enhance mitochondrial biogenesis demonstrates a direct pathway to ES cell differentiation and maturation into beating cardiomyocytes that express adult structural markers. Targeted HO-1/CO interventions up- and downregulate specific cardiogenic transcription factors, transcription factor Gata4, homeobox protein Nkx-2.5, heart- and neural crest derivatives-expressed protein 1, and MEF2C. HO-1/CO overexpression increases cardiac gene expression for myosin regulatory light chain 2, atrial isoform, MLC2v, ANP, MHC-β, and sarcomere α-actinin and the major mitochondrial fusion regulators, mitofusin 2 and MICOS complex subunit Mic60. This promotes structural mitochondrial network expansion and maturation, thereby supporting energy provision for beating embryoid bodies. These effects are prevented by silencing HO-1 and by mitochondrial reactive oxygen species scavenging, while disruption of mitochondrial biogenesis and mitochondrial DNA depletion by loss of mitochondrial transcription factor A compromise infrastructure. This leads to failure of cardiomyocyte differentiation and maturation and contractile dysfunction. INNOVATION: The capacity to augment cardiomyogenesis via a defined mitochondrial pathway has unique therapeutic potential for targeting ES cell maturation in cardiac disease. CONCLUSION: Our findings establish the HO-1/CO system and redox regulation of mitochondrial biogenesis as essential factors in ES cell differentiation as well as in the subsequent maturation of these cells into functional cardiac cells.
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    Implantation of mouse embryonic stem cell-derived cardiac progenitor cells preserves function of infarcted murine hearts.
    (PLoS One, 2010-07-12) Christoforou, Nicolas; Oskouei, Behzad N; Esteso, Paul; Hill, Christine M; Zimmet, Jeffrey M; Bian, Weining; Bursac, Nenad; Leong, Kam W; Hare, Joshua M; Gearhart, John D
    Stem cell transplantation holds great promise for the treatment of myocardial infarction injury. We recently described the embryonic stem cell-derived cardiac progenitor cells (CPCs) capable of differentiating into cardiomyocytes, vascular endothelium, and smooth muscle. In this study, we hypothesized that transplanted CPCs will preserve function of the infarcted heart by participating in both muscle replacement and neovascularization. Differentiated CPCs formed functional electromechanical junctions with cardiomyocytes in vitro and conducted action potentials over cm-scale distances. When transplanted into infarcted mouse hearts, CPCs engrafted long-term in the infarct zone and surrounding myocardium without causing teratomas or arrhythmias. The grafted cells differentiated into cross-striated cardiomyocytes forming gap junctions with the host cells, while also contributing to neovascularization. Serial echocardiography and pressure-volume catheterization demonstrated attenuated ventricular dilatation and preserved left ventricular fractional shortening, systolic and diastolic function. Our results demonstrate that CPCs can engraft, differentiate, and preserve the functional output of the infarcted heart.
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    Induced pluripotent stem cell-derived cardiac progenitors differentiate to cardiomyocytes and form biosynthetic tissues.
    (PLoS One, 2013) Christoforou, Nicolas; Liau, Brian; Chakraborty, Syandan; Chellapan, Malathi; Bursac, Nenad; Leong, Kam W
    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 drug development studies.
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    Mammalian genes induce partially reprogrammed pluripotent stem cells in non-mammalian vertebrate and invertebrate species.
    (Elife, 2013-09-03) Rosselló, Ricardo Antonio; Chen, Chun-Chun; Dai, Rui; Howard, Jason T; Hochgeschwender, Ute; Jarvis, Erich D
    Cells are fundamental units of life, but little is known about evolution of cell states. Induced pluripotent stem cells (iPSCs) are once differentiated cells that have been re-programmed to an embryonic stem cell-like state, providing a powerful platform for biology and medicine. However, they have been limited to a few mammalian species. Here we found that a set of four mammalian transcription factor genes used to generate iPSCs in mouse and humans can induce a partially reprogrammed pluripotent stem cell (PRPSCs) state in vertebrate and invertebrate model organisms, in mammals, birds, fish, and fly, which span 550 million years from a common ancestor. These findings are one of the first to show cross-lineage stem cell-like induction, and to generate pluripotent-like cells for several of these species with in vivo chimeras. We suggest that the stem-cell state may be highly conserved across a wide phylogenetic range. DOI:http://dx.doi.org/10.7554/eLife.00036.001.
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    The immune checkpoint molecule, VTCN1/B7-H4, guides differentiation and suppresses proinflammatory responses and MHC class I expression in an embryonic stem cell-derived model of human trophoblast.
    (Frontiers in endocrinology, 2023-01) Zhou, Jie; Tian, Yuchen; Qu, Ying; Williams, Madyson; Yuan, Ye; Karvas, Rowan M; Sheridan, Megan A; Schulz, Laura C; Ezashi, Toshihiko; Roberts, Michael R; Schust, Danny J
    The placenta acts as a protective barrier to pathogens and other harmful substances present in the maternal circulation throughout pregnancy. Disruption of placental development can lead to complications of pregnancy such as preeclampsia, intrauterine growth retardation and preterm birth. In previous work, we have shown that expression of the immune checkpoint regulator, B7-H4/VTCN1, is increased upon differentiation of human embryonic stem cells (hESC) to an in vitro model of primitive trophoblast (TB), that VTCN1/B7-H4 is expressed in first trimester but not term human placenta and that primitive trophoblast may be uniquely susceptible to certain pathogens. Here we report on the role of VTCN1 in trophoblast lineage development and anti-viral responses and the effects of changes in these processes on major histocompatibility complex (MHC) class I expression and peripheral NK cell phenotypes.
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    The secreted metalloprotease ADAMTS20 is required for melanoblast survival.
    (PLoS Genet, 2008-02-29) Silver, Debra L; Hou, Ling; Somerville, Robert; Young, Mary E; Apte, Suneel S; Pavan, William J
    ADAMTS20 (Adisintegrin-like and metalloprotease domain with thrombospondin type-1 motifs) is a member of a family of secreted metalloproteases that can process a variety of extracellular matrix (ECM) components and secreted molecules. Adamts20 mutations in belted (bt) mice cause white spotting of the dorsal and ventral torso, indicative of defective neural crest (NC)-derived melanoblast development. The expression pattern of Adamts20 in dermal mesenchymal cells adjacent to migrating melanoblasts led us to initially propose that Adamts20 regulated melanoblast migration. However, using a Dct-LacZ transgene to track melanoblast development, we determined that melanoblasts were distributed normally in whole mount E12.5 bt/bt embryos, but were specifically reduced in the trunk of E13.5 bt/bt embryos due to a seven-fold higher rate of apoptosis. The melanoblast defect was exacerbated in newborn skin and embryos from bt/bt animals that were also haploinsufficient for Adamts9, a close homolog of Adamts20, indicating that these metalloproteases functionally overlap in melanoblast development. We identified two potential mechanisms by which Adamts20 may regulate melanoblast survival. First, skin explant cultures demonstrated that Adamts20 was required for melanoblasts to respond to soluble Kit ligand (sKitl). In support of this requirement, bt/bt;Kit(tm1Alf)/+ and bt/bt;Kitl(Sl)/+ mice exhibited synergistically increased spotting. Second, ADAMTS20 cleaved the aggregating proteoglycan versican in vitro and was necessary for versican processing in vivo, raising the possibility that versican can participate in melanoblast development. These findings reveal previously unrecognized roles for Adamts proteases in cell survival and in mediating Kit signaling during melanoblast colonization of the skin. Our results have implications not only for understanding mechanisms of NC-derived melanoblast development but also provide insights on novel biological functions of secreted metalloproteases.
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    Tissue-engineered cardiac patch for advanced functional maturation of human ESC-derived cardiomyocytes.
    (Biomaterials, 2013-07) Zhang, Donghui; Shadrin, Ilya Y; Lam, Jason; Xian, Hai-Qian; Snodgrass, H Ralph; Bursac, Nenad
    Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) provide a promising source for cell therapy and drug screening. Several high-yield protocols exist for hESC-CM production; however, methods to significantly advance hESC-CM maturation are still lacking. Building on our previous experience with mouse ESC-CMs, we investigated the effects of 3-dimensional (3D) tissue-engineered culture environment and cardiomyocyte purity on structural and functional maturation of hESC-CMs. 2D monolayer and 3D fibrin-based cardiac patch cultures were generated using dissociated cells from differentiated Hes2 embryoid bodies containing varying percentage (48-90%) of CD172a (SIRPA)-positive cardiomyocytes. hESC-CMs within the patch were aligned uniformly by locally controlling the direction of passive tension. Compared to hESC-CMs in age (2 weeks) and purity (48-65%) matched 2D monolayers, hESC-CMs in 3D patches exhibited significantly higher conduction velocities (CVs), longer sarcomeres (2.09 ± 0.02 vs. 1.77 ± 0.01 μm), and enhanced expression of genes involved in cardiac contractile function, including cTnT, αMHC, CASQ2 and SERCA2. The CVs in cardiac patches increased with cardiomyocyte purity, reaching 25.1 cm/s in patches constructed with 90% hESC-CMs. Maximum contractile force amplitudes and active stresses of cardiac patches averaged to 3.0 ± 1.1 mN and 11.8 ± 4.5 mN/mm(2), respectively. Moreover, contractile force per input cardiomyocyte averaged to 5.7 ± 1.1 nN/cell and showed a negative correlation with hESC-CM purity. Finally, patches exhibited significant positive inotropy with isoproterenol administration (1.7 ± 0.3-fold force increase, EC50 = 95.1 nm). These results demonstrate highly advanced levels of hESC-CM maturation after 2 weeks of 3D cardiac patch culture and carry important implications for future drug development and cell therapy studies.
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    WNT3 is a biomarker capable of predicting the definitive endoderm differentiation potential of hESCs.
    (Stem Cell Reports, 2013) Jiang, Wei; Zhang, Donghui; Bursac, Nenad; Zhang, Yi
    Generation of functional cells from human pluripotent stem cells (PSCs) through in vitro differentiation is a promising approach for drug screening and cell therapy. However, the observed large and unavoidable variation in the differentiation potential of different human embryonic stem cell (hESC)/induced PSC (iPSC) lines makes the selection of an appropriate cell line for the differentiation of a particular cell lineage difficult. Here, we report identification of WNT3 as a biomarker capable of predicting definitive endoderm (DE) differentiation potential of hESCs. We show that the mRNA level of WNT3 in hESCs correlates with their DE differentiation efficiency. In addition, manipulations of hESCs through WNT3 knockdown or overexpression can respectively inhibit or promote DE differentiation in a WNT3 level-dependent manner. Finally, analysis of several hESC lines based on their WNT3 expression levels allowed accurate prediction of their DE differentiation potential. Collectively, our study supports the notion that WNT3 can serve as a biomarker for predicting DE differentiation potential of hESCs.