Optogenetic Stimulation of Gi Signaling Enables Instantaneous Modulation of Cardiomyocyte Pacemaking

G-protein signaling pathways are central in the regulation of cardiac function in physiological and pathophysiological conditions. Their functional analysis through optogenetic techniques with selective expression of opsin proteins and activation by specific wavelengths allows high spatial and temporal precision. Here, we present the application of long wavelength-sensitive cone opsin (LWO) in cardiomyocytes for activation of the Gi signaling pathway by red light. Murine embryonic stem (ES) cells expressing LWO were generated and differentiated into beating cardiomyocytes in embryoid bodies (EBs). Illumination with red light (625 nm) led to an instantaneous decrease up to complete inhibition (84–99% effectivity) of spontaneous beating, but had no effect on control EBs. By using increasing light intensities with 10 s pulses, we determined a half maximal effective light intensity of 2.4 μW/mm2 and a maximum effect at 100 μW/mm2. Pre-incubation of LWO EBs with pertussis toxin completely inhibited the light effect proving the specificity for Gi signaling. Frequency reduction was mainly due to the activation of GIRK channels because the specific channel blocker tertiapin reduced the light effect by ~80%. Compared with pharmacological stimulation of M2 receptors with carbachol with slow kinetics (>30 s), illumination of LWO had an identical efficacy, but much faster kinetics (<1 s) in the activation and deactivation demonstrating the temporal advantage of optogenetic stimulation. Thus, LWO is an effective optogenetic tool for selective stimulation of the Gi signaling cascade in cardiomyocytes with red light, providing high temporal precision.

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X chromosome retains the memory of its parental origin in murine embryonic stem cells

Development ◽

10.1242/dev.119.3.813 ◽

1993 ◽

Vol 119 (3) ◽

pp. 813-821 ◽

Cited By ~ 2

Author(s):

T. Tada ◽

M. Tada ◽

N. Takagi

Keyword(s):

X Chromosome ◽

Polar Region ◽

Biochemical Study ◽

Es Cells ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Parental Origin ◽

Visceral Endoderm ◽

Es Cell ◽

Preferential Inactivation

A cytogenetic and biochemical study of balloon-like cystic embryoid bodies, formed by newly established embryonic stem (ES) cell lines having a cytogenetically or genetically marked X chromosome, revealed that the paternally derived X chromosome was inactivated in the majority of cells in the yolk sac-like mural region consisting of the visceral endoderm and mesoderm. The nonrandomness was less evident in the more solid polar region containing the ectodermal vesicle, mesoderm and visceral endoderm. Since the same was true in embryoid bodies derived from ES cells at the 30th subculture generation, it was concluded that the imprinting responsible for the preferential inactivation of the paternal X chromosome that was limited to non-epiblast cells of the female mouse embryos, was stably maintained in undifferentiated ES cells. Differentiating epiblast cells should be able to erase or avoid responding to the imprint.

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Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro

Blood ◽

10.1182/blood.v87.7.2740.bloodjournal8772740 ◽

1996 ◽

Vol 87 (7) ◽

pp. 2740-2749 ◽

Cited By ~ 83

Author(s):

CD Helgason ◽

G Sauvageau ◽

HJ Lawrence ◽

C Largman ◽

RK Humphries

Keyword(s):

Stem Cells ◽

Es Cells ◽

Hematopoietic Cells ◽

Embryonic Stem ◽

Homeobox Genes ◽

Embryoid Bodies ◽

Proliferative Potential ◽

Hematopoietic Stem ◽

Differentiation And Proliferation

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

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Isolation, Characterization and Differentiation Potential of Chicken Spermatogonial Stem Cell Derived Embryoid Bodies

Annals of Animal Science ◽

10.1515/aoas-2015-0063 ◽

2016 ◽

Vol 16 (1) ◽

pp. 115-128 ◽

Cited By ~ 2

Author(s):

Thanh Luan Nguyen ◽

Jae Gyu Yoo ◽

Neelesh Sharma ◽

Sung Woo Kim ◽

Yong Jun Kang ◽

...

Keyword(s):

Developmental Biology ◽

Regenerative Medicine ◽

Connexin 43 ◽

Es Cells ◽

Periodic Acid ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Significant Finding ◽

Differentiation Potential

Abstract Human, murine and monkey spermatogonial stem cells (SSCs) have the capability to undergo self-renewal and differentiation into different body cell types in vitro, which are expected to serve as a powerful tool and resource for the developmental biology and regenerative medicine. We have successfully isolated and characterized the chicken SSCs from 3-day-old chicken testicular cells. The pluripotency was using Periodic Acid-Schiff (PAS ) staining or alkaline phosphatase staining, and antibodies to stage-specific embryonic antigens. In suspension culture conditions SSCs formed embryoid bodies (EBs) like embryonic stem (ES) cells. Subsequently EB differentiated into osteoblasts, adipocytes and most importantly into cardiomyocytes under induced differentiation conditions. The differentiation potential of EBs into cardiomyocyte-like cells was confirmed by using antibodies against sarcomeric α-actinin, cardiac troponin T and connexin 43. Cardiomyocytes-like cells were also confirmed by RT-PCR analysis for several cardiac cell genes like GATA-4, Nkx2-5, α-MHC, and ANF. We have successfully established an in vitro differentiation system for chicken SSCs into different body cells such as osteoblasts, adipocytes and cardiomyocytes. The most significant finding of this study is the differentiation potential of chicken SSCs into cardiomyocytes. Our findings may have implication in developmental biology and regenerative medicine by using chicken as the most potential animal model.

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Differentiating Embryonic Stem (ES) Cells into Embryoid Bodies

Cold Spring Harbor Protocols ◽

10.1101/pdb.prot4405 ◽

2006 ◽

Vol 2006 (2) ◽

pp. pdb.prot4405 ◽

Cited By ~ 2

Author(s):

Andras Nagy ◽

Marina Gertsenstein ◽

Kristina Vintersten ◽

Richard Behringer

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Embryoid Bodies

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Functional role of Tet-mediated RNA hydroxymethylcytosine in mouse ES cells and during differentiation

Nature Communications ◽

10.1038/s41467-020-18729-6 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Jie Lan ◽

Nicholas Rajan ◽

Martin Bizet ◽

Audrey Penning ◽

Nitesh K. Singh ◽

...

Keyword(s):

Es Cells ◽

Consensus Sequence ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Messenger Rnas ◽

Related Factors ◽

Mouse Es Cells ◽

Mrna Targets ◽

Tet Enzymes ◽

And Function

Abstract Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.

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Acceleration of mesoderm development and expansion of hematopoietic progenitors in differentiating ES cells by the mouse Mix-like homeodomain transcription factor

Blood ◽

10.1182/blood-2005-10-4120 ◽

2006 ◽

Vol 107 (8) ◽

pp. 3122-3130 ◽

Cited By ~ 29

Author(s):

Stephen Willey ◽

Angel Ayuso-Sacido ◽

Hailan Zhang ◽

Stuart T. Fraser ◽

Kenneth E. Sahr ◽

...

Keyword(s):

Es Cells ◽

Critical Role ◽

Embryonic Stem ◽

Homeobox Gene ◽

Embryoid Bodies ◽

Hematopoietic Progenitors ◽

Adult Type ◽

Mesoderm Development ◽

Molecular Events ◽

Organ Systems

Abstract The cellular and molecular events underlying the formation and differentiation of mesoderm to derivatives such as blood are critical to our understanding of the development and function of many tissues and organ systems. How different mesodermal populations are set aside to form specific lineages is not well understood. Although previous genetic studies in the mouse embryo have pointed to a critical role for the homeobox gene Mix-like (mMix) in gastrulation, its function in mesoderm development remains unclear. Hematopoietic defects have been identified in differentiating embryonic stem cells in which mMix was genetically inactivated. Here we show that conditional induction of mMix in embryonic stem cell–derived embryoid bodies results in the early activation of mesodermal markers prior to expression of Brachyury/T and acceleration of the mesodermal developmental program. Strikingly, increased numbers of mesodermal, hemangioblastic, and hematopoietic progenitors form in response to premature activation of mMix. Differentiation to primitive (embryonic) and definitive (adult type) blood cells proceeds normally and without an apparent bias in the representation of different hematopoietic cell fates. Therefore, the mouse Mix gene functions early in the recruitment and/or expansion of mesodermal progenitors to the hemangioblastic and hematopoietic lineages.

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Targeted Disruption of the Mouse Mitoferrin (Slc25A37) Mitochondrial Solute Carrier Results in Defective Primitive and Definitive Erythropoiesis.

Blood ◽

10.1182/blood.v108.11.265.265 ◽

2006 ◽

Vol 108 (11) ◽

pp. 265-265 ◽

Cited By ~ 3

Author(s):

Barry H. Paw ◽

Babette Gwynn ◽

Nathaniel B. Langer ◽

George C. Shaw ◽

Amy J. Lambert ◽

...

Keyword(s):

Aminolevulinic Acid ◽

Es Cells ◽

Embryonic Stem ◽

Embryoid Bodies ◽

Embryonic Lethality ◽

In Vitro Differentiation ◽

Mitochondrial Iron ◽

Solute Carrier ◽

Definitive Erythropoiesis

Abstract We previously described a zebrafish mutant, frascati (frs), which exhibits profound hypochromic anemia and erythroid maturation arrest due to defects in mitochondrial iron uptake. Through positional cloning, we showed that the frs gene encodes a novel member of the vertebrate mitochondrial solute carrier family (SLC25), mitoferrin (mfrn, slc25a37). Mfrn, which is highly expressed in fetal and adult hematopoietic tissues of zebrafish and mouse, functions as the major mitochondrial iron importer essential for heme biosynthesis in vertebrate erythroblasts (Shaw GC, et al. 2006 Nature 440:96–100). To study the function of Mfrn in mammalian organisms, we identified an embryonic stem (ES) cell clone that harbors a gene trap b-geo cassette in intron 1 that inactivates the Mfrn locus. Homozygous disruption of the Mfrn locus results in embryonic lethality at E11.5 from profound anemia due to a failure of primitive erythropoiesis, confirming the requirement of Mfrn in mammalian development . Circumventing the embryonic lethality, we generated Mfrn−/− ES cells to study the role of Mfrn in definitive erythropoiesis by in vitro differentiation of embryoid bodies and mixed chimera assays. Mfrn−/− ES cells were defective in promoting the growth, differentiation, and hemoglobinization of both primitive and definitive erythroblasts by in vitro differentiation of embryoid bodies. In mixed chimera studies, Mfrn−/− ES cells failed to contribute to the erythroid compartment of adult mosaic mice, whereas measurable contribution of Mfrn−/− donor cells could be assayed in the non-erythroid, leukocyte compartment. Transcriptome microarray analysis, using the mouse Affymetrix GeneChip and the custom IronChip, revealed unexpected down-regulation of transcripts for heme-biosynthetic enzymes in Mfrn−/− erythroblasts. The block in protoprophyrin synthesis, as well as mitochondrial heme synthesis, could be partially rescued by the addition of aminolevulinic acid (ALA) to Mfrn−/− erythroblasts in vitro. Our data demonstrate that mitochondrial iron homeostasis, working through the Mfrn iron importer, coordinately regulates the synthetic pathways for porphyrin and heme in developing mammalian erythroblasts.

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Specific Hematopoietic and Erythroid Differentiation Defects in Mouse Embryonic Stem (ES) Cells with Abortive Ribosome Assembly.

Blood ◽

10.1182/blood.v114.22.1088.1088 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1088-1088

Author(s):

Tracie A. Goldberg ◽

Adrianna Henson ◽

Sharon Singh ◽

Abdallah Nihrane ◽

Jeffrey Michael Lipton ◽

...

Keyword(s):

Cell Lines ◽

Colony Formation ◽

Conflicts Of Interest ◽

Es Cells ◽

Bone Marrow Failure ◽

Embryonic Stem ◽

Gene Trap ◽

Embryoid Bodies ◽

Ribosome Assembly ◽

Trap Cell

Abstract Abstract 1088 Poster Board I-110 Background Diamond Blackfan anemia (DBA) is one of the rare inherited bone marrow failure syndromes, characterized by erythroid hypoplasia, congenital anomalies and cancer predisposition. DBA has been shown to result from haploinsufficiency of ribosomal proteins (RPS19, RPS17, RPS24, RPL5, RPL11, RPL35a), which somehow triggers apoptosis of erythroid precursors. There is a marked variation in phenotype among members of the same family and also between subsets of patients with different mutations. Methods We studied primary and secondary in vitro differentiation of two murine ES gene trap cell lines with mutations in Rps19: S17-10H1, in which Rps19 is disrupted by insertion of the ROSAFARY gene trap vector between exons 2 and 3; and YHC074, in which the pGT0Lxf gene trap vector is inserted between exons 3 and 4 and whose growth is feeder cell-independent. For primary differentiation and generation of embryoid bodies (EBs), the ES cells were cultured in a serum-supplemented methylcellulose-based medium containing stem cell factor (SCF). After 7 days, the cultures were fed with a medium containing SCF, interleukin-3 (IL-3), IL-6 and erythropoietin (epo). EBs were scored on day 6 for total quantity, then again on day 13 for hematopoietic percentage. Secondary (hematopoietic) differentiation was performed on day 9 EBs. EBs were harvested and disrupted with collagenase, and the disrupted cells were suspended in a serum-supplemented methylcellulose-based medium with SCF, IL-3, IL-6 and epo. Hematopoietic colonies were counted on day 10. Results Decreased expression of Rps19 protein was confirmed by Western blot analysis in both S17-10H1 and YHC074 gene trap cell lines. We focused on YHC074 because its growth is feeder-independent, and it expresses approximately 50% of normal Rps19 levels. By polysome analysis, we found a selective reduction in the 40S subunit peak in mutant YHC074 cells as compared to parental controls. By Northern blot assays, we also found a relative increase in the 21S pre-rRNA to 18S rRNA ratio in mutant YHC074 cells. The viability of undifferentiated ES cells was not significantly different from parental control cells in the first 72 hours of culture; however, there was a significantly decreased number of EBs, particularly hematopoietic EBs, following primary differentiation (Fig. 1). Furthermore, when day 9 EBs were induced to secondary (hematopoietic) differentation, there was a significant decrease in the ratio of erythroid (CFU-E and BFU-E) to myeloid (CFU-GM) colony formation in mutant YHC074 cells. In order to confirm these results in an isogenic background, we stably transfected S17-10H1 cells with a vector expressing wild-type Rps19 cDNA and the puromycin resistance gene. Several resistant clones were found to overexpress Rps19 and were further studied in secondary differentiation experiments. There was a significant decrease in erythroid and myeloid colony formation and in BFU-E size from mutant S17-10H1 cells when compared to the Rps19-overexpressing clone, suggesting a direct relationship between the levels of Rps19 protein and hematopoietic growth and differentiation. Conclusion Using two ES cell lines with slightly different Rps19 mutations and genetic backgrounds, we have recapitulated the major DBA erythroid growth and differentiation defect, as well as the defect in ribosome assembly and rRNA processing caused by Rps19 haploinsufficiency. Disclosures No relevant conflicts of interest to declare.

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Filamin A Controls Embryonic Stem Cell Differentiation.

Blood ◽

10.1182/blood.v114.22.4599.4599 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4599-4599

Author(s):

Taisuke Kanaji ◽

Takashi Okamura ◽

Peter J. Newman

Keyword(s):

Cell Differentiation ◽

Signaling Pathways ◽

Es Cells ◽

Embryonic Stem ◽

Stem Cell Differentiation ◽

Embryoid Bodies ◽

Actin Binding ◽

Embryonic Stem Cell Differentiation ◽

Filamin A ◽

Es Cell

Abstract Abstract 4599 Filamin A is a major non-muscle actin binding protein that plays an important role in cross-linking cortical actin filaments into three-dimensional networks. In addition to its role as a cytoskeletal scaffolding molecule, Filamin A is also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. To analyze the role of filamin A in mouse embryonic stem (ES) cell maturation, we generated filamin ALow ES cells by introducing a micro-RNA that specifically downregulates filamin A expression under the control of a cytomegalovirus promoter. Filamin ALow ES cells exhibited a more rounded morphology than did their wild-type filamin ANormal counterparts, and expressed increased levels of the ES cell transcription factor Nanog. In contrast, non-transfected cells in the same culture dish retained normal expression of filamin A, expressed low levels of Nanog, and exhibited a more elongated and spread phenotype characteristic of differentiating cells. Further evidence for a role for filamin A in ES cell differentiation was provided by the observation that withdrawing leukemia inhibitory factor (LIF) to induce ES cell differentiation was accompanied by increased expression of filamin A, a concomitant loss of Nanog expression, and acquisition of a differentiated morphology. Filamin ALow ES cells were able to retain their undifferentiated phenotype, as evaluated by alkaline phosphatase (Alp) activity, in the presence of a 10-fold lower concentration of LIF than was permissive for filamin ANormal ES cells, or following exposure to the differentiating agent, bone morphogenic protein 4 (BMP4). LIF-induced phosphorylation of ERK was decreased in filamin ALow relative to filamin ANormal ES cells, as was BMP-induced phosphorylation of Smad1/5 - two signaling pathways that initiate ES cell differentiation. Finally, embryoid bodies comprised of filamin ALow ES cells were unable to differentiate into CD41+ hematopoietic progenitor cells. Taken together, these data demonstrate that filamin A plays a previously unrecognized, but critical, scaffolding function that support both the LIF - ERK and BMP4 - Smad1/5 signaling pathways leading to ES and hematopoietic cell differentiation. Manipulation of filamin levels might be useful in the future to modulate the differentiation requirements for a variety of clinically-and therapeutically-useful stem cells. Disclosures: Newman: Novo Nordisk: Consultancy; New York Blood Center: Membership on an entity's Board of Directors or advisory committees.

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Polarized and functional epithelia can form after the targeted inactivation of both mouse keratin 8 alleles.

The Journal of Cell Biology ◽

10.1083/jcb.115.6.1675 ◽

1991 ◽

Vol 115 (6) ◽

pp. 1675-1684 ◽

Cited By ~ 57

Author(s):

H Baribault ◽

R G Oshima

Keyword(s):

Es Cells ◽

Embryonic Stem ◽

Basal Membrane ◽

Embryoid Bodies ◽

Normal Epithelium ◽

Differentiated Cells ◽

Keratin 8 ◽

And Function ◽

Targeted Inactivation ◽

Keratin Intermediate Filaments

We have tested the requirement of keratin intermediate filaments for the formation and function of a simple epithelium. We disrupted both alleles of the mouse keratin 8 (mK8) gene in embryonic stem cells, and subsequently analyzed the phenotype in developing embryoid bodies in suspension culture. After the inactivation of the mouse keratin 8 (mK8) gene by a targeted insertion, mK8 protein synthesis was undetectable. In the absence of mK8 its complementary partners mK18 and mK19 were unable to form filaments within differentiated cells. Surprisingly, these ES cells differentiate to both simple and cystic embryoid bodies with apparently normal epithelia. Ultrastructural analysis shows an apparently normal epithelium with microvilli on the apical membrane, tight junctions and desmosomes on the lateral membrane, and an underlying basal membrane. No significant differences in the synthesis or secretion of alpha 1-fetoprotein and laminin were observed between the mK8- or wild-type embryoid bodies. Our data show that mK8 is not required for simple epithelium formation of extraembryonic endoderm.

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