Global transcriptional profiling reveals similarities and differences between human stem cell-derived cardiomyocyte clusters and heart tissue

2012 ◽  
Vol 44 (4) ◽  
pp. 245-258 ◽  
Author(s):  
Jane Synnergren ◽  
Caroline Améen ◽  
Andreas Jansson ◽  
Peter Sartipy
Keyword(s):  
Stem Cell ◽  
Ion Channels ◽  
Human Heart ◽  
Embryonic Stem ◽  
Heart Tissue ◽  
Phenotypic Characterization ◽  
Adult Heart ◽  
Promising Source

It is now well documented that human embryonic stem cells (hESCs) can differentiate into functional cardiomyocytes. These cells constitute a promising source of material for use in drug development, toxicity testing, and regenerative medicine. To assess their utility as replacement or complement to existing models, extensive phenotypic characterization of the cells is required. In the present study, we used microarrays and analyzed the global transcription of hESC-derived cardiomyocyte clusters (CMCs) and determined similarities as well as differences compared with reference samples from fetal and adult heart tissue. In addition, we performed a focused analysis of the expression of cardiac ion channels and genes involved in the Ca2+-handling machinery, which in previous studies have been shown to be immature in stem cell-derived cardiomyocytes. Our results show that hESC-derived CMCs, on a global level, have a highly similar gene expression profile compared with human heart tissue, and their transcriptional phenotype was more similar to fetal than to adult heart. Despite the high similarity to heart tissue, a number of significantly differentially expressed genes were identified, providing some clues toward understanding the molecular difference between in vivo sourced tissue and stem cell derivatives generated in vitro. Interestingly, some of the cardiac-related ion channels and Ca2+-handling genes showed differential expression between the CMCs and heart tissues. These genes may represent candidates for future genetic engineering to create hESC-derived CMCs that better mimic the phenotype of the cardiomyocytes present in the adult human heart.

scholarly journals Gene Expression Profiling of Functional Murine Embryonic Stem Cell-Derived Cardiomyocytes and Comparison with Adult Heart: Profiling of Murine ESC-Derived Cardiomyocytes

2009 ◽  
Vol 14 (3) ◽  
pp. 239-245 ◽  
Author(s):  
Tadahiro Shinozawa ◽  
Akiko Tsuji ◽  
Kenichi Imahashi ◽  
Kosuke Nakashima ◽  
Hiroshi Sawada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Ion Channels ◽  
Embryonic Stem Cell ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Heart Tissue ◽  
Embryoid Bodies ◽  
Efficient Production ◽  
Adult Heart

Although embryonic stem cell (ESC)—derived cardiomyocytes may be a powerful tool in drug discovery, their potential has not yet been fully explored. Nor has a detailed comparison with adult heart tissue been performed. We have developed a method for efficient production of cardiomyocyte-rich embryoid bodies (EBs) from murine ESCs. Analysis of global gene expression profiles showed that EBs on day 7 and/or 21 of differentiation (d7CMs and d21CMs, respectively) were similar to adult heart tissue for genes categorized as regulators of muscle contraction or voltage-gated ion channel activity, although d21CMs were more mature than d7CMs for contractile components related to morphological structures. Calcium and sodium channel blockers altered Ca2+ transients, and isoproterenol, a β-adrenergic compound, increased the rate of beating in d7CMs and d21CMs. Our gene analytic system therefore enabled us to identify genes that are expressed in the physiological pathways associated with ion channels and structural components in d7CMs and d21CMs. We conclude that EBs might be of use for the basic screening of drugs that might affect contractile function through ion channels. ( Journal of Biomolecular Screening 2009:239-245)

scholarly journals In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons

2005 ◽  
Vol 92 (5) ◽  
pp. 1265-1276 ◽  
Author(s):  
Chang-Hwan Park ◽  
Yang-Ki Minn ◽  
Ji-Yeon Lee ◽  
Dong Ho Choi ◽  
Mi-Yoon Chang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Dopamine Neurons

scholarly journals Znanstvena istraživanja u 21. stoljeću - etički aspekti uporabe laboratorijskih životinja i alternativni modeli

2022 ◽  
Vol 53 (5) ◽  
Author(s):  
Ivana Kmetič ◽  
Monika Roller ◽  
Marina Miletić ◽  
Teuta Murati
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryo Culture ◽  
Embryonic Stem ◽  
Whole Embryo Culture ◽  
Cell Test

U toksikološkim istraživanjima uz uporabu klasičnih (in vivo) istraživanja, primjenjuju se alternativni test sustavi. Korištenje laboratorijskih životinja, embrija, humanog i animalnog tkiva, kultura stanica i fetalnog seruma u istraživanjima smatra se etički problematičnim te se ograničava zakonima, pravilnicima i praksom. Razmatranjem načina kojima bi se neetičnost mogla izbjeći, došlo je do razvoja “3R” načela (akronim za tri pristupa koja bi se trebala provoditi pri istraživanjima na laboratorijskim životinjama), a to su: smanjenje/racionalizacija uporabe laboratorijskih životinja (engl. Reduction), načelo njihove zamjene (engl. Replacement) i poboljšanje uvjeta uzgoja, smještaja i skrbi za životinje (engl. Refinement). Većina je alternativnih testova toksičnosti još uvijek u postupku validacije. Pojedini in vitro testovi za istraživanja embriotoksičnosti (etički posebno osjetljivo područje) koja su priznala nadležna regulatorna tijela, su EST (engl. Embryonic Stem cell Test), WEC (engl. Whole- Embryo Culture) i MM (engl. MicroMass) test. Standardizacija protokola i uvođenje novih in vitro modela predstavlja važan segment napretka u toksikološkim istraživanjima. Znanstvena budućnost tu vidi mogućnost razvoja i implementacije načela etičnosti u istraživanja primjenjujući sustave koji će promišljeno i bez korištenja živih organizama dijelom nadomjestiti metode u biomedicini, veterinarskoj medicini, biotehnologiji i užem smislu - toksikologiji i farmakologiji.

Accept or Reject: The Role of Immune Tolerance in the Development of Stem Cell Therapies and Possible Future Approaches

2020 ◽  
pp. 019262332091824
Author(s):  
Richard Haworth ◽  
Michaela Sharpe
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem ◽  
Immune Recognition ◽  
Cell Of Origin ◽  
In Vivo Models ◽  
Cell Product ◽  
A Cell

In 2011, Goldring and colleagues published a review article describing the potential safety issues of novel stem cell-derived treatments. Immunogenicity and immunotoxicity of the administered cell product were considered risks in the light of clinical experience of transplantation. The relative immunogenicity of mesenchymal stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) was being addressed through in vitro and in vivo models. But the question arose as to whether the implanted cells needed to be identical to the recipient in every respect, including epigenetically, to evade immune recognition? If so, this set a high bar which may preclude use of many cells derived from iPSCs which have vestiges of a fetal phenotype and epigenetic memory of their cell of origin. However, for autologous iPSCs, the immunogenicity reduces once the surface antigen expression profile becomes close to that of the parent somatic cells. Therefore, a cell product containing incompletely differentiated cells could be more immunogenic. The properties of the administered cells, the immune privilege of the administration site, and the host immune status influence graft success or failure. In addition, the various approaches available to characterize potential immunogenicity of a cell therapy will be discussed.

scholarly journals Quantitative Analysis of Dopamine Neuron Subtypes Generated from Mouse Embryonic Stem Cells

2016 ◽  
Author(s):  
Yu-Ting L. Dingle ◽  
Katherine B. Xiong ◽  
Jason T. Machan ◽  
Kimberly A. Seymour ◽  
Debra Ellisor ◽  
...  
Keyword(s):  
Stem Cell ◽  
Sonic Hedgehog ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Systematic Random Sampling ◽  
Brain Science ◽  
Fibroblast Growth Factor 8 ◽  
Brown University

AbstractDopamine (DA) neuron subtypes modulate specific physiological functions and are involved in distinct neurological disorders. Embryonic stem cell (ESC) derived DA neurons have the potential to aid in the study of disease mechanisms, drug discovery, and possibly cell replacement therapies. DA neurons can be generated from ESCs in vitro, but the subtypes of ESC-derived DA neurons have not been investigated in detail despite the diversity of DA neurons observed in vivo. Due to cell culture heterogeneity, sampling methods applied to ESC-derived cultures can be ambiguous and potentially biased. Therefore, we developed a quantification method to capture the depth of DA neuron production in vitro by estimating the error associated with systematic random sampling. Using this method, we quantified calbindin+ and calretinin+ subtypes of DA neurons generated from mouse ESCs. We found a higher production of the calbindin+ subtype (11−27%) compared to the calretinin+ subtype (2-13%) of DA neuron; in addition, DA neurons expressing neither subtype marker were also generated. We then examined whether exogenous sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) affected subtype generation. Our results demonstrate that exogenous SHH and FGF8 did not alter DA neuron subtype generation in vitro. These findings suggest that a deeper understanding DA neuron derivation inclusive of mechanisms that govern the in vitro subtype specification of ESC-derived DA neurons is required.NoteAll research was planned and conducted while members were at Brown UniversityResearch fundingNIH/NCRR/NIGMS RI Hospital COBRE Center for Stem Cell Biology (8P20GM103468-04) (MZ) Brown Institute for Brain Science Pilot Grant (4-63662) (MZ/DHK)

scholarly journals Fezf2 transient expression via modRNA with concurrent SIRT1 inhibition enhances differentiation of cortical subcerebral / corticospinal neuron identity from mES cells

2020 ◽  
Author(s):  
Cameron Sadegh ◽  
Wataru Ebina ◽  
Anthony C. Arvanites ◽  
Lance S. Davidow ◽  
Lee L. Rubin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cortical Neurons ◽  
Embryonic Stem ◽  
Induced Pluripotent Stem Cell ◽  
Sirtuin 1 ◽  
Projection Neurons ◽  
Specific Expression ◽  
Specific Differentiation

AbstractDuring late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

scholarly journals In Vitro Generation of Oocyte Like Cells and Their In Vivo Efficacy: How Far We have been Succeeded

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 557 ◽  
Author(s):  
Dinesh Bharti ◽  
Si-Jung Jang ◽  
Sang-Yun Lee ◽  
Sung-Lim Lee ◽  
Gyu-Jin Rho
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem ◽  
In Vivo Studies ◽  
Special Focus ◽  
Morphological Alterations ◽  
Cell Sources ◽  
Induced Pluripotent

In the last few decades, stem cell therapy has grown as a boon for many pathological complications including female reproductive disorders. In this review, a brief description of available strategies that are related to stem cell-based in vitro oocyte-like cell (OLC) development are given. We have tried to cover all the aspects and latest updates of the in vitro OLC developmental methodologies, marker profiling, available disease models, and in vivo efficacies, with a special focus on mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs) usage. The differentiation abilities of both the ovarian and non-ovarian stem cell sources under various induction conditions have shown different effects on morphological alterations, proliferation- and size-associated developments, hormonal secretions under gonadotropic stimulations, and their neo-oogenesis or folliculogenesis abilities after in vivo transplantations. The attainment of characters like oocyte-like morphology, size expansion, and meiosis initiation have been found to be major obstacles during in vitro oogenesis. A number of reports have either lacked in vivo studies or have shown their functional incapability to produce viable and healthy offspring. Though researchers have gained many valuable insights regarding in vitro gametogenesis, still there are many things to do to make stem cell-derived OLCs fully functional.

182 Establishment of expanded potential embryonic stem cell lines from porcine embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 215
Author(s):  
M. Nowak-Imialek ◽  
X. Gao ◽  
P. Liu ◽  
H. Niemann
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Germ Layers ◽  
Embryonic Tissues

The domestic pig is an excellent large animal in biomedical medicine and holds great potential for testing the clinical safety and efficacy of stem cell therapies. Previously, numerous studies reported the derivation of porcine embryonic stem cell (ESC)-like lines, but none of these lines fulfilled the stringent criteria for true pluripotent germline competent ESC. Here, we report the first establishment of porcine expanded potential stem cells (pEPSC) from parthenogenetic and in vivo-derived blastocysts. A total of 12 cell lines from parthenogenetic blastocysts from Day 7 (12/24) and 26 cell lines from in vivo-derived blastocysts from Day 5 (26/27) were established using defined stem cell culture conditions. These cells closely resembled mouse ESC with regard to morphology, formed compact colonies with high nuclear/cytoplasmic ratios, and could be maintained in vitro for more than 40 passages with a normal karyotype. The pEPSC expressed key pluripotency genes, including OCT4, NANOG, SOX2, and SALL4 at similar levels as porcine blastocysts. Immunostaining analysis confirmed expression of critical cell surface markers SSEA-1 and SSEA-4 in pEPSC. The EPSC differentiated in vitro into tissues expressing markers of the 3 germ layers: SOX7, AFP, T, DES, CRABP2, α-SMA, β-tubulin, PAX6, and, notably, the trophoblast markers HAND1, GATA3, PGF, and KRT7. After injection into immunocompromised mice, the pEPSC formed teratomas with derivatives of the 3 germ layers and placental lactogen-1 (PL-1)-positive trophoblast-like cells. Additionally, pEPSC cultured in vitro under conditions specific for germ cells formed embryoid bodies, which contained ~9% primordial germ cell (PGC)-like cells (PGCLC) that expressed PGC-specific genes, including NANOS3, BLIMP1, TFAP2C, CD38, DND1, KIT, and OCT4 as detected by quantitative RT-PCR and immunostaining. Next, we examined the in vivo differentiation potential of pEPSC and injected pEPSC stably expressing the CAG-H2B-mCherry transgene reporter into porcine embryos. The donor cells proliferated and were localised in both the trophectoderm and inner cell mass of the blastocysts cultured in vitro. After transfer to 3 recipient sows, chimeric embryos implanted and a total of 45 fetuses were recovered on Days 26 to 28. Flow cytometry of single cells collected from embryonic and extraembryonic tissues of the fetuses revealed mCherry+ cells in 7 conceptuses, in both the placenta and embryonic tissues; in 3 chimeric conceptuses, mCherry+ cells were exclusively found in embryonic tissues; and in 2 conceptuses, mCherry+ cells were exclusively localised in the placenta. The contribution of the mCherry+ cells was low (0.4-1.7%), but they were found and co-detected in multiple porcine embryonic tissues using tissue lineage-specific markers, including SOX2, TUJ1, GATA4, SOX17, AFP, α-SMA, and trophoblast markers PL-1 and KRT7 in the placental cells. The successful establishment of pEPSC represents a major step forward in stem cell research and provides cell lines with the unique state of cellular potency useful for genetic engineering and unravelling pluripotency regulation in pigs.

Abstract 78: Pnmt+ "Primer" Cells Give Rise to Cardiac Myocytes and Neurons in the Mammalian Heart: Development of New Experimental Tools for Cardiac Regenerative Medicine Applications

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Namita M Varudkar ◽  
Jixiang Xia ◽  
Ibrahim Abukenda ◽  
Karl Pfeifer ◽  
Steven Ebert
Keyword(s):  
Stem Cell ◽  
Regenerative Medicine ◽  
Neural Crest ◽  
Heart Development ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Stem Cell Differentiation ◽  
Left Ventricular

Phenylethanolamine n-methyltransferase (Pnmt) catalyzes the conversion of norepinephrine to epinephrine, and thus serves as a marker for adrenergic cells. We employed a combination of immunofluorescent histochemical staining and genetic fate-mapping strategies to show that two separate Pnmt+ cell populations contribute to heart development. Intrinsic cardiac adrenergic (ICA) cells originate from the primary heart field, and contribute to pacemaking, conduction, and working (contractile) myocardium. A second population of cardiac Pnmt+ cells is derived from migrating neural crest. These neural crest adrenergic (NCA) cells appear to contribute to cardiac neurons. By adulthood, most of the Pnmt+ cells show a distinctively left-sided orientation in the heart, with nearly 90% of them being found in the left atrium and ventricle. Surprisingly large swaths of ventricular muscle are derived from Pnmt+ primer cells. Since this region of the heart is highly vulnerable to coronary artery disease and often sustains varying degrees of damage following myocardial infarction, we hypothesize that directed stem cell differentiation into Pnmt+ primer cells could serve as a valuable resource for repair and/or regeneration of left ventricular myocardium for heart disease patients. To test this hypothesis, we have generated stable recombinant mouse embryonic stem cell (mESC) lines that express various fluorescent marker proteins under the control of the endogenous Pnmt gene regulatory network. These cells can be rapidly expanded in culture, sorted, and used for transplantation studies in animal models to determine their therapeutic effectiveness. The cells can be induced along cardiogenic or neurogenic pathways in vitro, and the resulting Pnmt+ cells from each population can then be collected and tested in vivo. To achieve this goal, we have knocked-in a nuclear-localized enhanced green fluorescent protein into the Pnmt locus to create Pnmt-nEGFP recombinant mESCs and mice. We show that nEGFP expression is specifically expressed in Pnmt+ cells in vitro and in vivo. This strategy allows us to identify and isolate Pnmt+ cells to evaluate their effectiveness for cardiac regenerative medicine applications. .

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