Publications by authors named "Janet Rossant"

219 Publications

Tryptophan-metabolizing gut microbes regulate adult neurogenesis via the aryl hydrocarbon receptor.

Proc Natl Acad Sci U S A 2021 Jul;118(27)

Lee Kong Chian School of Medicine, Nanyang Technological University, Singapore 636921;

While modulatory effects of gut microbes on neurological phenotypes have been reported, the mechanisms remain largely unknown. Here, we demonstrate that indole, a tryptophan metabolite produced by tryptophanase-expressing gut microbes, elicits neurogenic effects in the adult mouse hippocampus. Neurogenesis is reduced in germ-free (GF) mice and in GF mice monocolonized with a single-gene knockout (KO) mutant unable to produce indole. External administration of systemic indole increases adult neurogenesis in the dentate gyrus in these mouse models and in specific pathogen-free (SPF) control mice. Indole-treated mice display elevated synaptic markers postsynaptic density protein 95 and synaptophysin, suggesting synaptic maturation effects in vivo. By contrast, neurogenesis is not induced by indole in aryl hydrocarbon receptor KO (AhR) mice or in ex vivo neurospheres derived from them. Neural progenitor cells exposed to indole exit the cell cycle, terminally differentiate, and mature into neurons that display longer and more branched neurites. These effects are not observed with kynurenine, another AhR ligand. The indole-AhR-mediated signaling pathway elevated the expression of , , and genes, thus identifying a molecular pathway connecting gut microbiota composition and their metabolic function to neurogenesis in the adult hippocampus. Our data have implications for the understanding of mechanisms of brain aging and for potential next-generation therapeutic opportunities.
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http://dx.doi.org/10.1073/pnas.2021091118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8271728PMC
July 2021

ISSCR Guidelines for Stem Cell Research and Clinical Translation: The 2021 update.

Stem Cell Reports 2021 Jun 27;16(6):1398-1408. Epub 2021 May 27.

Histogen, San Diego, CA, USA.

The International Society for Stem Cell Research has updated its Guidelines for Stem Cell Research and Clinical Translation in order to address advances in stem cell science and other relevant fields, together with the associated ethical, social, and policy issues that have arisen since the last update in 2016. While growing to encompass the evolving science, clinical applications of stem cells, and the increasingly complex implications of stem cell research for society, the basic principles underlying the Guidelines remain unchanged, and they will continue to serve as the standard for the field and as a resource for scientists, regulators, funders, physicians, and members of the public, including patients. A summary of the key updates and issues is presented here.
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http://dx.doi.org/10.1016/j.stemcr.2021.05.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190668PMC
June 2021

Human embryo research, stem cell-derived embryo models and in vitro gametogenesis: Considerations leading to the revised ISSCR guidelines.

Stem Cell Reports 2021 Jun 27;16(6):1416-1424. Epub 2021 May 27.

Gairdner Foundation, Toronto, Ontario, Canada. Electronic address:

The ISSCR Guidelines for Stem Cell Research and Clinical Translation were last revised in 2016. Since then, rapid progress has been made in research areas related to in vitro culture of human embryos, creation of stem cell-based embryo models, and in vitro gametogenesis. Therefore, a working group of international experts was convened to review the oversight process and provide an update to the guidelines. This report captures the discussion and summarizes the major recommendations made by this working group, with a specific emphasis on updating the categories of review and engagement with the specialized scientific and ethical oversight process.
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http://dx.doi.org/10.1016/j.stemcr.2021.05.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8190666PMC
June 2021

Fetal lung underdevelopment is rescued by administration of amniotic fluid stem cell extracellular vesicles in rodents.

Sci Transl Med 2021 04;13(590)

Developmental and Stem Cell Biology Program, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, M5G 0A4, Canada.

Fetal lung underdevelopment, also known as pulmonary hypoplasia, is characterized by decreased lung growth and maturation. The most common birth defect found in babies with pulmonary hypoplasia is congenital diaphragmatic hernia (CDH). Despite research and clinical advances, babies with CDH still have high morbidity and mortality rates, which are directly related to the severity of lung underdevelopment. To date, there is no effective treatment that promotes fetal lung growth and maturation. Here, we describe a stem cell-based approach in rodents that enhances fetal lung development via the administration of extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs). Using fetal rodent models of pulmonary hypoplasia (primary epithelial cells, organoids, explants, and in vivo), we demonstrated that AFSC-EV administration promoted branching morphogenesis and alveolarization, rescued tissue homeostasis, and stimulated epithelial cell and fibroblast differentiation. We confirmed this regenerative ability in in vitro models of lung injury using human material, where human AFSC-EVs obtained following good manufacturing practices restored pulmonary epithelial homeostasis. Investigating EV mechanism of action, we found that AFSC-EV beneficial effects were exerted via the release of RNA cargo. MicroRNAs regulating the expression of genes involved in lung development, such as the miR17-92 cluster and its paralogs, were highly enriched in AFSC-EVs and were increased in AFSC-EV-treated primary lung epithelial cells compared to untreated cells. Our findings suggest that AFSC-EVs hold regenerative ability for underdeveloped fetal lungs, demonstrating potential for therapeutic application in patients with pulmonary hypoplasia.
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http://dx.doi.org/10.1126/scitranslmed.aax5941DOI Listing
April 2021

Opportunities and challenges with stem cell-based embryo models.

Stem Cell Reports 2021 May 4;16(5):1031-1038. Epub 2021 Mar 4.

Children's Medical Research Institute, University of Sydney, and School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Sydney, Australia. Electronic address:

Stem cell-based embryo models open an unprecedented avenue for modeling embryogenesis, cell lineage differentiation, tissue morphogenesis, and organogenesis in mammalian development. Experimentation on these embryo models can lead to a better understanding of the mechanisms of development and offers opportunities for functional genomic studies of disease-causing mechanisms, identification of therapeutic targets, and preclinical modeling of advanced therapeutics for precision medicine. An immediate challenge is to create embryo models of high fidelity to embryogenesis and organogenesis in vivo, to ensure that the knowledge gleaned is biologically meaningful and clinically relevant.
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http://dx.doi.org/10.1016/j.stemcr.2021.02.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8185371PMC
May 2021

Evaluating totipotency using criteria of increasing stringency.

Nat Cell Biol 2021 01 8;23(1):49-60. Epub 2021 Jan 8.

Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.

Totipotency is the ability of a single cell to give rise to all of the differentiated cell types that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies on a variety of assays of variable stringency. Here, we describe criteria to define totipotency. We explain how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in mice, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbour increased totipotent potential relative to conventional embryonic stem cells under in vitro and in vivo conditions.
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http://dx.doi.org/10.1038/s41556-020-00609-2DOI Listing
January 2021

Canada Gairdner Awards 2020: A Tightly Woven Tapestry of Discovery.

Authors:
Janet Rossant

Cell 2020 04 31;181(3):507-508. Epub 2020 Mar 31.

President and Scientific Director, The Gairdner Foundation, Toronto, ON, Canada. Electronic address:

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http://dx.doi.org/10.1016/j.cell.2020.03.027DOI Listing
April 2020

Toward Guidelines for Research on Human Embryo Models Formed from Stem Cells.

Stem Cell Reports 2020 02 16;14(2):169-174. Epub 2020 Jan 16.

Hospital for Sick Children and the Department of Molecular Genetics, University of Toronto, Toronto, Canada.

Over the past few years, a number of research groups have reported striking progress on the generation of in vitro models from mouse and human stem cells that replicate aspects of early embryonic development. Not only do these models reproduce some key cell fate decisions but, especially in the mouse system, they also mimic the spatiotemporal arrangements of embryonic and extraembryonic tissues that are required for developmental patterning and implantation in the uterus. If such models could be developed for the early human embryo, they would have great potential benefits for understanding early human development, for biomedical science, and for reducing the use of animals and human embryos in research. However, guidelines for the ethical conduct of this line of work are at present not well defined. In this Forum article, we discuss some key aspects of this emerging area of research and provide some recommendations for its ethical oversight.
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http://dx.doi.org/10.1016/j.stemcr.2019.12.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7015820PMC
February 2020

Efficient Generation of Large-Fragment Knock-In Mouse Models Using 2-Cell (2C)-Homologous Recombination (HR)-CRISPR.

Curr Protoc Mouse Biol 2020 Mar;10(1):e67

Program in Developmental and Stem Cell Biology, Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, Toronto, Ontario, Canada.

Generating large-fragment knock-ins, such as reporters, conditional alleles, or humanized alleles, directly in mouse embryos is still a challenging feat. We have developed 2C-HR-CRISPR, a technology that allows highly efficient (10-50%) and rapid (generating founders in 2 months) targeting of large DNA fragments. Key to this strategy is the delivery of CRISPR reagents into 2-cell-stage mouse embryos, taking advantage of the high homologous recombination activity during the long G cell cycle phase at this stage. Furthermore, by exploiting a Cas9-monomeric streptavidin (Cas-mSA) and biotinylated PCR template (BioPCR) system to localize the repair template to specific double strand breaks, the efficiency can be further improved to up to 95%. Here we provide a procedure to generate large-fragment knock-in mouse models using 2C-HR-CRISPR. We first describe the principles for designing single guide RNAs and repair templates but refer to published manuscripts and protocols for molecular cloning methods or commercial sources for these reagents. We then describe two unique aspects of 2C-HR-CRISPR that are critical for success: (1) production of the CRISPR reagents for 2C-HR-CRISPR, particularly for applying the Cas9-mSA/BioPCR method, and (2) microinjection of mouse embryos at the 2-cell stage. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Single guide RNA and repair template design Basic Protocol 2: Preparing reagents for 2C-HR-CRISPR Basic Protocol 3: Microinjecting 2-cell-stage mouse embryos.
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http://dx.doi.org/10.1002/cpmo.67DOI Listing
March 2020

Esrrb function is required for proper primordial germ cell development in presomite stage mouse embryos.

Dev Biol 2019 11 14;455(2):382-392. Epub 2019 Jul 14.

Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. Electronic address:

Estrogen related receptor beta (Esrrb) is an orphan nuclear receptor that is required for self-renewal and pluripotency in mouse embryonic stem (ES) cells. However, in the early post-implantation mouse embryo, Esrrb is specifically expressed in the extraembryonic ectoderm (ExE) and plays a crucial role in trophoblast development. Previous studies showed that Esrrb is also required to maintain trophoblast stem (TS) cells, the in vitro stem cell model of the early trophoblast lineage. In order to identify regulatory targets of Esrrb in vivo, we performed microarray analysis of Esrrb-null versus wild-type post-implantation ExE, and identified 30 genes down-regulated in Esrrb-mutants. Among them is Bmp4, which is produced by the ExE and known to be critical for primordial germ cell (PGC) specification in vivo. We further identified an enhancer region bound by Esrrb at the Bmp4 locus by performing Esrrb ChIP-seq and luciferase reporter assay using TS cells. Finally, we established a knockout mouse line in which the enhancer region was deleted using CRISPR/Cas9 technology. Both Esrrb-null embryos and enhancer knockout embryos expressed lower levels of Bmp4 in the ExE, and had reduced numbers of PGCs. These results suggested that Esrrb functions as an upstream factor of Bmp4 in the ExE, regulating proper PGC development in mice.
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http://dx.doi.org/10.1016/j.ydbio.2019.07.008DOI Listing
November 2019

Inhibition of Phosphoinositide-3-Kinase Signaling Promotes the Stem Cell State of Trophoblast.

Stem Cells 2019 10 18;37(10):1307-1318. Epub 2019 Jul 18.

Centre for Trophoblast Research, University of Cambridge, Cambridge, United Kingdom.

Trophoblast stem cells (TSCs) are a heterogeneous cell population despite the presence of fibroblast growth factor (FGF) and transforming growth factor β (TGFB) as key growth factors in standard culture conditions. To understand what other signaling cascades control the stem cell state of mouse TSCs, we performed a kinase inhibitor screen and identified several novel pathways that cause TSC differentiation. Surprisingly, inhibition of phosphoinositide-3-kinase (PI3K) signaling increased the mRNA and protein expression of stem cell markers instead, and resulted in a tighter epithelial colony morphology and fewer differentiated cells. PI3K inhibition could not substitute for FGF or TGFB and did not affect phosphorylation of extracellular signal-regulated kinase, and thus acts independently of these pathways. Upon removal of PI3K inhibition, TSC transcription factor levels reverted to normal TSC levels, indicating that murine TSCs can reversibly switch between these two states. In summary, PI3K inhibition reduces the heterogeneity and seemingly heightens the stem cell state of TSCs as indicated by the simultaneous upregulation of multiple key marker genes and cell morphology. Stem Cells 2019;37:1307-1318.
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http://dx.doi.org/10.1002/stem.3052DOI Listing
October 2019

Conversion of human and mouse fibroblasts into lung-like epithelial cells.

Sci Rep 2019 06 21;9(1):9027. Epub 2019 Jun 21.

Program in Developmental & Stem Cell Biology, SickKids Research Institute, Hospital for Sick Children, Toronto, ON, Canada.

Cell lineage conversion of fibroblasts to specialized cell types through transdifferentiation may provide a fast and alternative cell source for regenerative medicine. Here we show that transient transduction of fibroblasts with the four reprogramming factors (Oct4, Sox2, Klf4, and c-Myc) in addition to the early lung transcription factor Nkx2-1 (also known as Ttf1), followed by directed differentiation of the cells, can convert mouse embryonic and human adult dermal fibroblasts into induced lung-like epithelial cells (iLEC). These iLEC differentiate into multiple lung cell types in air liquid interface cultures, repopulate decellularized rat lung scaffolds, and form lung epithelia composed of Ciliated, Goblet, Basal, and Club cells after transplantation into immune-compromised mice. As proof-of-concept, differentiated human iLEC harboring the Cystic Fibrosis mutation dF508 demonstrated pharmacological rescue of CFTR function using the combination of lumacaftor and ivacaftor. Overall, this is a promising alternative approach for generation of patient-specific lung-like progenitors to study lung function, disease and future regeneration strategies.
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http://dx.doi.org/10.1038/s41598-019-45195-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6588580PMC
June 2019

2018 ISSCR Strategic Planning: Looking to the Future.

Stem Cell Reports 2019 06;12(6):1183-1185

Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, ON, Canada; Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada; The Gairdner Foundation, Toronto, ON, Canada. Electronic address:

In 2018, the ISSCR underwent a strategic planning process to identify current priorities and plan for future initiatives. Key recommendations will help the society better serve its members and fulfill its mission of promoting excellence in stem cell science and applications to human health.
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http://dx.doi.org/10.1016/j.stemcr.2019.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6566000PMC
June 2019

Preparation of Polymerase Chain Reaction Templates from Embryonic and Adult Mouse Tissue Samples.

Cold Spring Harb Protoc 2019 03 1;2019(3). Epub 2019 Mar 1.

A simple cell or tissue lysate can provide a sufficient quality and amount of template DNA for polymerase chain reaction (PCR). This protocol can be used to prepare embryonic tissues, yolk sac, ear punch, toe, or 1- to 2-mm (maximum 5 mm) tail samples for PCR analysis.
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http://dx.doi.org/10.1101/pdb.prot094391DOI Listing
March 2019

Genetic Control of Early Cell Lineages in the Mammalian Embryo.

Authors:
Janet Rossant

Annu Rev Genet 2018 11 5;52:185-201. Epub 2018 Sep 5.

Program in Stem Cell and Developmental Biology, Hospital for Sick Children, Toronto, Ontario M5G 0A4, Canada; email:

Establishing the different lineages of the early mammalian embryo takes place over several days and several rounds of cell divisions from the fertilized egg. The resulting blastocyst contains the pluripotent cells of the epiblast, from which embryonic stem cells can be derived, as well as the extraembryonic lineages required for a mammalian embryo to survive in the uterine environment. The dynamics of the cellular and genetic interactions controlling the initiation and maintenance of these lineages in the mouse embryo are increasingly well understood through application of the tools of single-cell genomics, gene editing, and in vivo imaging. Exploring the similarities and differences between mouse and human development will be essential for translation of these findings into new insights into human biology, derivation of stem cells, and improvements in fertility treatments.
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http://dx.doi.org/10.1146/annurev-genet-120116-024544DOI Listing
November 2018

Gene editing in human development: ethical concerns and practical applications.

Authors:
Janet Rossant

Development 2018 07 25;145(16). Epub 2018 Jul 25.

Peter Gilgan Centre for Research and Learning, Hospital for Sick Children, and Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5G 0A4

The amazing power of CRISPR-Cas9 gene editing tools and other related technologies has impacted all areas of biology today. It has also raised ethical concerns, particularly with regard to the possibility of generating heritable changes in the human genome - so-called germline gene editing. Although technical and safety issues suggest that this approach is far from clinical application, gene editing as a research tool is moving forward in human embryos, non-human primates and in stem cell-derived embryoids. These studies are already providing new information relevant to our understanding of normal human development, infertility, early pregnancy loss and pluripotent stem cell origins.
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http://dx.doi.org/10.1242/dev.150888DOI Listing
July 2018

Efficient generation of targeted large insertions by microinjection into two-cell-stage mouse embryos.

Nat Biotechnol 2018 08 11;36(7):632-637. Epub 2018 Jun 11.

Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.

Rapid, efficient generation of knock-in mice with targeted large insertions remains a major hurdle in mouse genetics. Here, we describe two-cell homologous recombination (2C-HR)-CRISPR, a highly efficient gene-editing method based on introducing CRISPR reagents into embryos at the two-cell stage, which takes advantage of the open chromatin structure and the likely increase in homologous-recombination efficiency during the long G2 phase. Combining 2C-HR-CRISPR with a modified biotin-streptavidin approach to localize repair templates to target sites, we achieved a more-than-tenfold increase (up to 95%) in knock-in efficiency over standard methods. We targeted 20 endogenous genes expressed in blastocysts with fluorescent reporters and generated reporter mouse lines. We also generated triple-color blastocysts with all three lineages differentially labeled, as well as embryos carrying the two-component auxin-inducible degradation system for probing protein function. We suggest that 2C-HR-CRISPR is superior to random transgenesis or standard genome-editing protocols, because it ensures highly efficient insertions at endogenous loci and defined 'safe harbor' sites.
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http://dx.doi.org/10.1038/nbt.4166DOI Listing
August 2018

Exploring early human embryo development.

Science 2018 06;360(6393):1075-1076

Embryology Unit, Children's Medical Research Institute and School of Medical Sciences, Faculty of Medicine and Health, University of Sydney, Westmead, NSW 2145, Australia.

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http://dx.doi.org/10.1126/science.aas9302DOI Listing
June 2018

The CF Canada-Sick Kids Program in individual CF therapy: A resource for the advancement of personalized medicine in CF.

J Cyst Fibros 2019 01 10;18(1):35-43. Epub 2018 Oct 10.

Molecular Structure and Function, Hospital for Sick Children, Toronto, Canada; Department of Physiology, University of Toronto, Toronto, Canada; Department of Biochemistry, University of Toronto, Toronto, Canada. Electronic address:

Background: Therapies targeting certain CFTR mutants have been approved, yet variations in clinical response highlight the need for in-vitro and genetic tools that predict patient-specific clinical outcomes. Toward this goal, the CF Canada-Sick Kids Program in Individual CF Therapy (CFIT) is generating a "first of its kind", comprehensive resource containing patient-specific cell cultures and data from 100 CF individuals that will enable modeling of therapeutic responses.

Methods: The CFIT program is generating: 1) nasal cells from drug naïve patients suitable for culture and the study of drug responses in vitro, 2) matched gene expression data obtained by sequencing the RNA from the primary nasal tissue, 3) whole genome sequencing of blood derived DNA from each of the 100 participants, 4) induced pluripotent stem cells (iPSCs) generated from each participant's blood sample, 5) CRISPR-edited isogenic control iPSC lines and 6) prospective clinical data from patients treated with CF modulators.

Results: To date, we have recruited 57 of 100 individuals to CFIT, most of whom are homozygous for F508del (to assess in-vitro: in-vivo correlations with respect to ORKAMBI response) or heterozygous for F508del and a minimal function mutation. In addition, several donors are homozygous for rare nonsense and missense mutations. Nasal epithelial cell cultures and matched iPSC lines are available for many of these donors.

Conclusions: This accessible resource will enable development of tools that predict individual outcomes to current and emerging modulators targeting F508del-CFTR and facilitate therapy discovery for rare CF causing mutations.
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http://dx.doi.org/10.1016/j.jcf.2018.03.013DOI Listing
January 2019

Modeling signaling-dependent pluripotency with Boolean logic to predict cell fate transitions.

Mol Syst Biol 2018 01 29;14(1):e7952. Epub 2018 Jan 29.

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON, Canada

Pluripotent stem cells (PSCs) exist in multiple stable states, each with specific cellular properties and molecular signatures. The mechanisms that maintain pluripotency, or that cause its destabilization to initiate development, are complex and incompletely understood. We have developed a model to predict stabilized PSC gene regulatory network (GRN) states in response to input signals. Our strategy used random asynchronous Boolean simulations (R-ABS) to simulate single-cell fate transitions and strongly connected components (SCCs) strategy to represent population heterogeneity. This framework was applied to a reverse-engineered and curated core GRN for mouse embryonic stem cells (mESCs) and used to simulate cellular responses to combinations of five signaling pathways. Our simulations predicted experimentally verified cell population compositions and input signal combinations controlling specific cell fate transitions. Extending the model to PSC differentiation, we predicted a combination of signaling activators and inhibitors that efficiently and robustly generated a Cdx2Oct4 cells from naïve mESCs. Overall, this platform provides new strategies to simulate cell fate transitions and the heterogeneity that typically occurs during development and differentiation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5787708PMC
http://dx.doi.org/10.15252/msb.20177952DOI Listing
January 2018

The role of Cdx2 as a lineage specific transcriptional repressor for pluripotent network during the first developmental cell lineage segregation.

Sci Rep 2017 12 7;7(1):17156. Epub 2017 Dec 7.

Center for Stem Cell and Regenerative Medicine, Zhejiang University School of Medicine, Hangzhou, 310058, China.

The first cellular differentiation event in mouse development leads to the formation of the blastocyst consisting of the inner cell mass (ICM) and trophectoderm (TE). The transcription factor CDX2 is required for proper TE specification, where it promotes expression of TE genes, and represses expression of Pou5f1 (OCT4). However its downstream network in the developing embryo is not fully characterized. Here, we performed high-throughput single embryo qPCR analysis in Cdx2 null embryos to identify CDX2-regulated targets in vivo. To identify genes likely to be regulated by CDX2 directly, we performed CDX2 ChIP-Seq on trophoblast stem (TS) cells. In addition, we examined the dynamics of gene expression changes using inducible CDX2 embryonic stem (ES) cells, so that we could predict which CDX2-bound genes are activated or repressed by CDX2 binding. By integrating these data with observations of chromatin modifications, we identify putative novel regulatory elements that repress gene expression in a lineage-specific manner. Interestingly, we found CDX2 binding sites within regulatory elements of key pluripotent genes such as Pou5f1 and Nanog, pointing to the existence of a novel mechanism by which CDX2 maintains repression of OCT4 in trophoblast. Our study proposes a general mechanism in regulating lineage segregation during mammalian development.
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http://dx.doi.org/10.1038/s41598-017-16009-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5719399PMC
December 2017

Cardiac-enriched BAF chromatin-remodeling complex subunit Baf60c regulates gene expression programs essential for heart development and function.

Biol Open 2018 Jan 5;7(1). Epub 2018 Jan 5.

Gladstone Institutes, San Francisco, CA, 94158 USA

How chromatin-remodeling complexes modulate gene networks to control organ-specific properties is not well understood. For example, () encodes a cardiac-enriched subunit of the SWI/SNF-like BAF chromatin complex, but its role in heart development is not fully understood. We found that constitutive loss of leads to embryonic cardiac hypoplasia and pronounced cardiac dysfunction. Conditional deletion of in cardiomyocytes resulted in postnatal dilated cardiomyopathy with impaired contractile function. regulates a gene expression program that includes genes encoding contractile proteins, modulators of sarcomere function, and cardiac metabolic genes. Many of the genes deregulated in null embryos are targets of the MEF2/SRF co-factor Myocardin (MYOCD). In a yeast two-hybrid screen, we identified MYOCD as a BAF60c interacting factor; we showed that BAF60c and MYOCD directly and functionally interact. We conclude that Baf60c is essential for coordinating a program of gene expression that regulates the fundamental functional properties of cardiomyocytes.
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http://dx.doi.org/10.1242/bio.029512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5829499PMC
January 2018

Regulation of porcupine-dependent Wnt signaling is essential for uterine development and function.

Reproduction 2018 01 24;155(1):93-102. Epub 2017 Oct 24.

McGill University Health CentreMontreal, Quebec, Canada

Six members of the Wnt family are expressed in the female reproductive tract. Their collective function ensures proper development of the uterus, preparing it for pregnancy during adulthood. Here, we take advantage of the fact that a prerequisite for all Wnt secretion, is located on the X chromosome, to generate females that were mosaic for throughout the reproductive tract. females were mated with progesterone receptor ()-Cre males ( ) to generate females that were heterozygous for Porcupine in all tissues of the female reproductive tract, resulting in mosaicism due to random X-inactivation. We demonstrated that mosaic females are extremely subfertile and exhibit a large spectrum of phenotypes ranging from morphologically normal uteri to uteri with extremely enlarged cystic glands. Decreased fertility in Porcupine mosaic females was not associated with phenotype severity and was observed regardless of whether or not cystic glands were enlarged. By crossing-in a GFP reporter on the wild-type X chromosome, we were able to correlate endometrial gland hyperplasia with a mostly mutant stroma, demonstrating the role of stromal Wnts in the regulation of endometrial gland proliferation. Finally, we demonstrated that fertility issues within mosaic females were due to a reduced response to estrogen and to abnormal Tcf/Lef signaling across the mesometrial-anti-mesometrial axis during the window of implantation.
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http://dx.doi.org/10.1530/REP-17-0436DOI Listing
January 2018

Porcupine-dependent Wnt activity within the uterine epithelium is essential for fertility.

Biol Reprod 2017 Nov;97(5):688-697

Department of Obstetrics and Gynecology, McGill University Health Centre, Montreal, Quebec, Canada.

The secretion of mammalian Wnt ligands within the cell is dependent on the activity of Porcupine, a gene located on the X-chromosome that encodes for a membrane-bound O-acyl transferase. Here, we report that postnatal ablation of Porcupine in the uterine luminal epithelium alone results in the decrease in endometrial gland number. Despite having uterine glands, mutant females are completely infertile. Epithelial ablation of Porcupine causes defects in timely apposition of the lumen, along with failure to respond to artificial decidual induction. Interestingly, progesterone supplementation was able to rescue the initiation of decidualization, but the decidua was not maintained and subsequently resorbed. Transcriptome analysis demonstrated that deletion of Porcupine in the epithelium resulted in the stromal dysregulation of members of the Wnt signaling pathway (Lef1, Wnt4, and Wnt16), dysregulation of receptors and ligands in the Notch signaling pathway (Notch1, Notch4, and Dll4) as well as Hoxa10. Our results demonstrate the crucial requirement of Wnt signaling in the epithelium for fertility and demonstrate that epithelial Wnts regulate stromal Wnt gene expression as well as regulating the expression of essential signaling factors and effectors required for successful embryo implantation.
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http://dx.doi.org/10.1093/biolre/iox119DOI Listing
November 2017

Human Embryo Editing: Opportunities and Importance of Transnational Cooperation.

Cell Stem Cell 2017 10;21(4):423-426

California Institute of Technology, Pasadena, CA 91125, USA; Organizing Committee for the International Summit on Human Gene Editing.

A recent National Academies report articulates a path forward for research, ethics, and governance of clinical applications involving genome editing. In light of recent human embryo editing developments, scientists and stakeholders from all nations should cooperate to take advantage of this historic opportunity for medicine and also basic human biology.
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http://dx.doi.org/10.1016/j.stem.2017.09.010DOI Listing
October 2017

AIRE is a critical spindle-associated protein in embryonic stem cells.

Elife 2017 07 25;6. Epub 2017 Jul 25.

Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada.

Embryonic stem (ES) cells go though embryo-like cell cycles regulated by specialized molecular mechanisms. However, it is not known whether there are ES cell-specific mechanisms regulating mitotic fidelity. Here we showed that Autoimmune Regulator (), a transcription coordinator involved in immune tolerance processes, is a critical spindle-associated protein in mouse ES(mES) cells. BioID analysis showed that AIRE associates with spindle-associated proteins in mES cells. Loss of function analysis revealed that was important for centrosome number regulation and spindle pole integrity specifically in mES cells. We also identified the c-terminal LESLL motif as a critical motif for AIRE's mitotic function. Combined maternal and zygotic knockout further revealed Aire's critical functions for spindle assembly in preimplantation embryos. These results uncovered a previously unappreciated function for and provide new insights into the biology of stem cell proliferation and potential new angles to understand fertility defects in humans carrying e mutations.
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http://dx.doi.org/10.7554/eLife.28131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5560860PMC
July 2017

Phenotypic profiling of CFTR modulators in patient-derived respiratory epithelia.

NPJ Genom Med 2017 Apr;2:12

Department of Physiology, University of Toronto, Toronto, ON, Canada.

Pulmonary disease is the major cause of morbidity and mortality in patients with cystic fibrosis, a disease caused by mutations in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. Heterogeneity in CFTR genotype-phenotype relationships in affected individuals plus the escalation of drug discovery targeting specific mutations highlights the need to develop robust in vitro platforms with which to stratify therapeutic options using relevant tissue. Toward this goal, we adapted a fluorescence plate reader assay of apical CFTR-mediated chloride conductance to enable profiling of a panel of modulators on primary nasal epithelial cultures derived from patients bearing different CFTR mutations. This platform faithfully recapitulated patient-specific responses previously observed in the "gold-standard" but relatively low-throughput Ussing chamber. Moreover, using this approach, we identified a novel strategy with which to augment the response to an approved drug in specific patients. In proof of concept studies, we also validated the use of this platform in measuring drug responses in lung cultures differentiated from cystic fibrosis iPS cells. Taken together, we show that this medium throughput assay of CFTR activity has the potential to stratify cystic fibrosis patient-specific responses to approved drugs and investigational compounds in vitro in primary and iPS cell-derived airway cultures.
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http://dx.doi.org/10.1038/s41525-017-0015-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5481189PMC
April 2017

Position- and Hippo signaling-dependent plasticity during lineage segregation in the early mouse embryo.

Elife 2017 02 22;6. Epub 2017 Feb 22.

Program in Developmental and Stem Cell Biology, Hospital for Sick Children, Toronto, Canada.

The segregation of the trophectoderm (TE) from the inner cell mass (ICM) in the mouse blastocyst is determined by position-dependent Hippo signaling. However, the window of responsiveness to Hippo signaling, the exact timing of lineage commitment and the overall relationship between cell commitment and global gene expression changes are still unclear. Single-cell RNA sequencing during lineage segregation revealed that the TE transcriptional profile stabilizes earlier than the ICM and prior to blastocyst formation. Using quantitative Cdx2-eGFP expression as a readout of Hippo signaling activity, we assessed the experimental potential of individual blastomeres based on their level of Cdx2-eGFP expression and correlated potential with gene expression dynamics. We find that TE specification and commitment coincide and occur at the time of transcriptional stabilization, whereas ICM cells still retain the ability to regenerate TE up to the early blastocyst stage. Plasticity of both lineages is coincident with their window of sensitivity to Hippo signaling.
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http://dx.doi.org/10.7554/eLife.22906DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5370188PMC
February 2017

New Insights into Early Human Development: Lessons for Stem Cell Derivation and Differentiation.

Cell Stem Cell 2017 01;20(1):18-28

Embryology Unit, Children's Medical Research Institute and School of Medical Sciences, Sydney Medical School, University of Sydney, Westmead, NSW 2145, Australia. Electronic address:

Pathways underlying mouse embryonic development have always informed efforts to derive, maintain, and drive differentiation of human pluripotent stem cells. However, direct application of mouse embryology to the human system has not always been successful because of fundamental developmental differences between species. The naive pluripotent state of mouse embryonic stem cells (ESCs), in particular, has been difficult to capture in human ESCs, and appears to be transitory in the human embryo itself. Further studies of human and non-human primate embryo development are needed to untangle the complexities of pluripotency networks across mammalian species.
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http://dx.doi.org/10.1016/j.stem.2016.12.004DOI Listing
January 2017
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