Publications by authors named "Jacob H Hanna"

58 Publications

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis.

Mol Cell 2020 12 19;80(5):876-891.e6. Epub 2020 Nov 19.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel. Electronic address:

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molcel.2020.10.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7816607PMC
December 2020

Context-dependent functional compensation between Ythdf mA reader proteins.

Genes Dev 2020 Oct 17;34(19-20):1373-1391. Epub 2020 Sep 17.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.

The N6-methyladenosine (mA) modification is the most prevalent post-transcriptional mRNA modification, regulating mRNA decay and splicing. It plays a major role during normal development, differentiation, and disease progression. The modification is regulated by a set of writer, eraser, and reader proteins. The YTH domain family of proteins consists of three homologous mA-binding proteins, Ythdf1, Ythdf2, and Ythdf3, which were suggested to have different cellular functions. However, their sequence similarity and their tendency to bind the same targets suggest that they may have overlapping roles. We systematically knocked out (KO) the Mettl3 writer, each of the Ythdf readers, and the three readers together (triple-KO). We then estimated the effect in vivo in mouse gametogenesis, postnatal viability, and in vitro in mouse embryonic stem cells (mESCs). In gametogenesis, severity is increased as the deletion occurs earlier in the process, and Ythdf2 has a dominant role that cannot be compensated by Ythdf1 or Ythdf3, due to differences in readers' expression pattern across different cell types, both in quantity and in spatial location. Knocking out the three readers together and systematically testing viable offspring genotypes revealed a redundancy in the readers' role during early development that is gene dosage-dependent. Finally, in mESCs there is compensation between the three Ythdf reader proteins, since the resistance to differentiate and the significant effect on mRNA decay occur only in the triple-KO cells and not in the single KOs. Thus, we suggest a new model for the Ythdf readers function, in which there is profound dosage-dependent redundancy when all three readers are equivalently coexpressed in the same cell types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.340695.120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7528697PMC
October 2020

Production and Analysis of Human Primordial Germ Cell-Like Cells.

Methods Mol Biol 2021 ;2195:125-145

Center for Cancer Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, USA.

Primordial germ cells (PGCs) are common ancestors of all germline cells. In mammals, PGCs emerge in early-stage embryos around the timing of gastrulation at or near epiblast, and specification of PGCs from their precursor cells involves multiple growth factors secreted by adjacent cells. Recent advancements in germline stem cell biology have made it possible to generate PGC-like cell culture models (PGCLCs for PGC-like cells) from human and mouse pluripotent stem cells by mimicking the embryonic growth factor environment in vitro. Here we describe a method of producing human PGCLCs from primed-pluripotency induced pluripotent stem cells (iPSCs) via temporal conversion to naive pluripotency followed by formation of embryoid bodies (EBs) using the spin-EB method.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/978-1-0716-0860-9_10DOI Listing
January 2021

β-Catenin safeguards the ground state of mousepluripotency by strengthening the robustness of the transcriptional apparatus.

Sci Adv 2020 Jul 17;6(29):eaba1593. Epub 2020 Jul 17.

Laboratory of Integrative Biology, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that β-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/sciadv.aba1593DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439582PMC
July 2020

Generation of human endothelium in pig embryos deficient in ETV2.

Nat Biotechnol 2020 03 24;38(3):297-302. Epub 2020 Feb 24.

Department of Medicine, University of Minnesota, Minneapolis, MN, USA.

The scarcity of donor organs may be addressed in the future by using pigs to grow humanized organs with lower potential for immunological rejection after transplantation in humans. Previous studies have demonstrated that interspecies complementation of rodent blastocysts lacking a developmental regulatory gene can generate xenogeneic pancreas and kidney. However, such organs contain host endothelium, a source of immune rejection. We used gene editing and somatic cell nuclear transfer to engineer porcine embryos deficient in ETV2, a master regulator of hematoendothelial lineages. ETV2-null pig embryos lacked hematoendothelial lineages and were embryonic lethal. Blastocyst complementation with wild-type porcine blastomeres generated viable chimeric embryos whose hematoendothelial cells were entirely donor-derived. ETV2-null blastocysts were injected with human induced pluripotent stem cells (hiPSCs) or hiPSCs overexpressing the antiapoptotic factor BCL2, transferred to synchronized gilts and analyzed between embryonic day 17 and embryonic day 18. In these embryos, all endothelial cells were of human origin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41587-019-0373-yDOI Listing
March 2020

Deciphering the "mA Code" via Antibody-Independent Quantitative Profiling.

Cell 2019 07 27;178(3):731-747.e16. Epub 2019 Jun 27.

Department of Molecular Genetics, Weizmann Institute of Science, 7610001 Rehovot, Israel. Electronic address:

N6-methyladenosine (mA) is the most abundant modification on mRNA and is implicated in critical roles in development, physiology, and disease. A major limitation has been the inability to quantify mA stoichiometry and the lack of antibody-independent methodologies for interrogating mA. Here, we develop MAZTER-seq for systematic quantitative profiling of m6A at single-nucleotide resolution at 16%-25% of expressed sites, building on differential cleavage by an RNase. MAZTER-seq permits validation and de novo discovery of mA sites, calibration of the performance of antibody-based approaches, and quantitative tracking of mA dynamics in yeast gametogenesis and mammalian differentiation. We discover that m6A stoichiometry is "hard coded" in cis via a simple and predictable code, accounting for 33%-46% of the variability in methylation levels and allowing accurate prediction of mA loss and acquisition events across evolution. MAZTER-seq allows quantitative investigation of mA regulation in subcellular fractions, diverse cell types, and disease states.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2019.06.013DOI Listing
July 2019

Stage-specific requirement for Mettl3-dependent mA mRNA methylation during haematopoietic stem cell differentiation.

Nat Cell Biol 2019 06 6;21(6):700-709. Epub 2019 May 6.

Columbia Stem Cell Initiative, Department of Rehabilitation and Regenerative Medicine and Department of Microbiology and Immunology, Columbia University Medical Center, New York, NY, USA.

Haematopoietic stem cells (HSCs) maintain balanced self-renewal and differentiation, but how these functions are precisely regulated is not fully understood. N-methyladenosine (mA) messenger RNA methylation has emerged as an important mode of epitranscriptional gene expression regulation affecting many biological processes. We show that deletion of the mA methyltransferase Mettl3 from the adult haematopoietic system led to an accumulation of HSCs in the bone marrow and a marked reduction of reconstitution potential due to a blockage of HSC differentiation. Interestingly, deleting Mettl3 from myeloid cells using Lysm-cre did not impact myeloid cell number or function. RNA sequencing revealed 2,073 genes with significant mA modifications in HSCs. Myc was identified as a direct target of mA in HSCs. Mettl3-deficient HSCs failed to upregulate MYC expression following stimulation to differentiate and enforced expression of Myc rescued differentiation defects of Mettl3-deficient HSCs. Our results reveal a key role of mA in governing HSC differentiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41556-019-0318-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6556891PMC
June 2019

Universally non-immunogenic iPSCs.

Nat Biomed Eng 2019 05;3(5):337-338

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41551-019-0401-8DOI Listing
May 2019

Stem Cell-Derived Human Gametes: The Public Engagement Imperative.

Trends Mol Med 2019 03 6;25(3):165-167. Epub 2019 Feb 6.

Department of Developmental Stem Cell Biology, Faculty of Medical Sciences, Kyushu University, Fukuoka, Japan.

The implications of scientific breakthroughs are rarely faced up to in advance of their realization. Stem cell-derived human gametes, a disruptive technology in waiting, are likely to recapitulate this historic pattern absent active intervention. Herein we call for the conduct of thoughtful ante hoc deliberations on the prospect of stem cell-derived human gametes with an eye toward minimizing potential untoward post hoc regulatory or statutory impositions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.molmed.2019.01.005DOI Listing
March 2019

Generation of Human Primordial Germ Cell-like Cells at the Surface of Embryoid Bodies from Primed-pluripotency Induced Pluripotent Stem Cells.

J Vis Exp 2019 01 11(143). Epub 2019 Jan 11.

Center for Cancer Research, Massachusetts General Hospital;

Primordial germ cells (PGCs) are common precursors of all germline cells. In mouse embryos, a founding population of ~40 PGCs are induced from pluripotent epiblast cells by orchestrated exposures to cytokines, including bone morphogenetic protein 4 (Bmp4). In human embryos, the earliest PGCs have been identified on the endodermal wall of yolk sac around the end of the 3 week of gestation, but little is known about the process of human PGC specification and their early development. To circumvent the technical and ethical barriers of studying human embryonic PGCs, surrogate cell culture models have been recently generated from pluripotent stem cells. Here, we describe a 13-day protocol for robust production of human PGC-Like Cells (hPGCLCs). Human induced pluripotent stem cells (hiPSCs) maintained in the primed pluripotency state are incubated in the 4i naïve reprogramming medium for 48 hours, dissociated to single cells, and packed into microwells. Prolonged maintenance of hiPSCs in the naïve pluripotency state causes significant chromosomal aberrations and should be avoided. hiPSCs in the microwells are maintained for an additional 24 hours in the 4i medium to form embryoid bodies (EBs), which are then cultured in low-adherence plasticware under a rocking condition in the hPGCLC induction medium containing a high concentration of recombinant human BMP4. EBs are further cultured for up to 8 days in the rocking, non-adherent condition to obtain maximum yields of hPGCLCs. By immunohistochemistry, hPGCLCs are readily detected as cells strongly expressing OCT4 in almost all EBs exclusively on their surface. When EBs are enzymatically dissociated and subjected to FACS enrichment, hPGCLCs can be collected as CD38+ cells with up to 40-45% yield.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3791/58297DOI Listing
January 2019

Publisher Correction: m6A modification controls the innate immune response to infection by targeting type I interferons.

Nat Immunol 2019 02;20(2):243

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

In the version of this article initially published, the penultimate sentence of the abstract included a typographical error ('cxgenes'). The correct word is 'genes'. The error has been corrected in the HTML and PDF version of the article.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41590-019-0314-4DOI Listing
February 2019

The N-Methyladenosine mRNA Methylase METTL3 Controls Cardiac Homeostasis and Hypertrophy.

Circulation 2019 01;139(4):533-545

Department of Physiology and Cell Biology (L.E.D., F.A.), Dorothy M. Davis Heart and Lung Research Institute, The Ohio State University, Columbus.

Background: N-Methyladenosine (m6A) methylation is the most prevalent internal posttranscriptional modification on mammalian mRNA. The role of m6A mRNA methylation in the heart is not known.

Methods: To determine the role of m6A methylation in the heart, we isolated primary cardiomyocytes and performed m6A immunoprecipitation followed by RNA sequencing. We then generated genetic tools to modulate m6A levels in cardiomyocytes by manipulating the levels of the m6A RNA methylase methyltransferase-like 3 (METTL3) both in culture and in vivo. We generated cardiac-restricted gain- and loss-of-function mouse models to allow assessment of the METTL3-m6A pathway in cardiac homeostasis and function.

Results: We measured the level of m6A methylation on cardiomyocyte mRNA, and found a significant increase in response to hypertrophic stimulation, suggesting a potential role for m6A methylation in the development of cardiomyocyte hypertrophy. Analysis of m6A methylation showed significant enrichment in genes that regulate kinases and intracellular signaling pathways. Inhibition of METTL3 completely abrogated the ability of cardiomyocytes to undergo hypertrophy when stimulated to grow, whereas increased expression of the m6A RNA methylase METTL3 was sufficient to promote cardiomyocyte hypertrophy both in vitro and in vivo. Finally, cardiac-specific METTL3 knockout mice exhibit morphological and functional signs of heart failure with aging and stress, showing the necessity of RNA methylation for the maintenance of cardiac homeostasis.

Conclusions: Our study identified METTL3-mediated methylation of mRNA on N-adenosines as a dynamic modification that is enhanced in response to hypertrophic stimuli and is necessary for a normal hypertrophic response in cardiomyocytes. Enhanced m6A RNA methylation results in compensated cardiac hypertrophy, whereas diminished m6A drives eccentric cardiomyocyte remodeling and dysfunction, highlighting the critical importance of this novel stress-response mechanism in the heart for maintaining normal cardiac function.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/CIRCULATIONAHA.118.036146DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340720PMC
January 2019

mA modification controls the innate immune response to infection by targeting type I interferons.

Nat Immunol 2019 02 17;20(2):173-182. Epub 2018 Dec 17.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel.

N-methyladenosine (mA) is the most common mRNA modification. Recent studies have revealed that depletion of mA machinery leads to alterations in the propagation of diverse viruses. These effects were proposed to be mediated through dysregulated methylation of viral RNA. Here we show that following viral infection or stimulation of cells with an inactivated virus, deletion of the mA 'writer' METTL3 or 'reader' YTHDF2 led to an increase in the induction of interferon-stimulated genes. Consequently, propagation of different viruses was suppressed in an interferon-signaling-dependent manner. Significantly, the mRNA of IFNB, the gene encoding the main cytokine that drives the type I interferon response, was mA modified and was stabilized following repression of METTL3 or YTHDF2. Furthermore, we show that mA-mediated regulation of interferon genes was conserved in mice. Together, our findings uncover the role mA serves as a negative regulator of interferon response by dictating the fast turnover of interferon mRNAs and consequently facilitating viral propagation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41590-018-0275-zDOI Listing
February 2019

Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules.

Cell Stem Cell 2019 02 13;24(2):328-341.e9. Epub 2018 Dec 13.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 761001, Israel. Electronic address:

The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2018.11.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116520PMC
February 2019

MTCH2-mediated mitochondrial fusion drives exit from naïve pluripotency in embryonic stem cells.

Nat Commun 2018 12 3;9(1):5132. Epub 2018 Dec 3.

Department of Biological Regulation, Weizmann Institute of Science, 7610001, Rehovot, Israel.

The role of mitochondria dynamics and its molecular regulators remains largely unknown during naïve-to-primed pluripotent cell interconversion. Here we report that mitochondrial MTCH2 is a regulator of mitochondrial fusion, essential for the naïve-to-primed interconversion of murine embryonic stem cells (ESCs). During this interconversion, wild-type ESCs elongate their mitochondria and slightly alter their glutamine utilization. In contrast, MTCH2 ESCs fail to elongate their mitochondria and to alter their metabolism, maintaining high levels of histone acetylation and expression of naïve pluripotency markers. Importantly, enforced mitochondria elongation by the pro-fusion protein Mitofusin (MFN) 2 or by a dominant negative form of the pro-fission protein dynamin-related protein (DRP) 1 is sufficient to drive the exit from naïve pluripotency of both MTCH2 and wild-type ESCs. Taken together, our data indicate that mitochondria elongation, governed by MTCH2, plays a critical role and constitutes an early driving force in the naïve-to-primed pluripotency interconversion of murine ESCs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-018-07519-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277412PMC
December 2018

Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency.

Cell Stem Cell 2018 09 16;23(3):412-425.e10. Epub 2018 Aug 16.

Department of Molecular Genetics, Weizmann Institute of Science, 234 Herzl, Rehovot 76100, Israel. Electronic address:

Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity. We identify Gatad2a, a NuRD-specific subunit, whose complete deletion specifically disrupts Mbd3/NuRD repressive activity on the pluripotency circuitry during iPSC differentiation and reprogramming without ablating somatic cell proliferation. Inhibition of Gatad2a facilitates deterministic murine iPSC reprogramming within 8 days. We validate a distinct molecular axis, Gatad2a-Chd4-Mbd3, within Mbd3/NuRD as being critical for blocking reestablishment of naive pluripotency and further highlight signaling-dependent and post-translational modifications of Mbd3/NuRD that influence its interactions and assembly.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stem.2018.07.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7116536PMC
September 2018

The Role of mA/m-RNA Methylation in Stress Response Regulation.

Neuron 2018 07;99(2):389-403.e9

Department of Stress Neurobiology and Neurogenetics, Max Planck Institute of Psychiatry, Munich 80804, Germany; Department of Neurobiology, Weizmann Institute of Science, Rehovot 76100, Israel. Electronic address:

N-methyladenosine (mA) and N,2'-O-dimethyladenosine (mAm) are abundant mRNA modifications that regulate transcript processing and translation. The role of both, here termed mA/m, in the stress response in the adult brain in vivo is currently unknown. Here, we provide a detailed analysis of the stress epitranscriptome using mA/m-seq, global and gene-specific mA/m measurements. We show that stress exposure and glucocorticoids region and time specifically alter mA/m and its regulatory network. We demonstrate that deletion of the methyltransferase Mettl3 or the demethylase Fto in adult neurons alters the mA/m epitranscriptome, increases fear memory, and changes the transcriptome response to fear and synaptic plasticity. Moreover, we report that regulation of mA/m is impaired in major depressive disorder patients following glucocorticoid stimulation. Our findings indicate that brain mA/m represents a novel layer of complexity in gene expression regulation after stress and that dysregulation of the mA/m response may contribute to the pathophysiology of stress-related psychiatric disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.neuron.2018.07.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6069762PMC
July 2018

Modulating cell state to enhance suspension expansion of human pluripotent stem cells.

Proc Natl Acad Sci U S A 2018 06 4;115(25):6369-6374. Epub 2018 Jun 4.

Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, ON M5S 3G9, Canada;

The development of cell-based therapies to replace missing or damaged tissues within the body or generate cells with a unique biological activity requires a reliable and accessible source of cells. Human pluripotent stem cells (hPSC) have emerged as a strong candidate cell source capable of extended propagation in vitro and differentiation to clinically relevant cell types. However, the application of hPSC in cell-based therapies requires overcoming yield limitations in large-scale hPSC manufacturing. We explored methods to convert hPSC to alternative states of pluripotency with advantageous bioprocessing properties, identifying a suspension-based small-molecule and cytokine combination that supports increased single-cell survival efficiency, faster growth rates, higher densities, and greater expansion than control hPSC cultures. ERK inhibition was found to be essential for conversion to this altered state, but once converted, ERK inhibition led to a loss of pluripotent phenotype in suspension. The resulting suspension medium formulation enabled hPSC suspension yields 5.7 ± 0.2-fold greater than conventional hPSC in 6 d, for at least five passages. Treated cells remained pluripotent, karyotypically normal, and capable of differentiating into all germ layers. Treated cells could also be integrated into directed differentiated strategies as demonstrated by the generation of pancreatic progenitors (NKX6.1+/PDX1+ cells). Enhanced suspension-yield hPSC displayed higher oxidative metabolism and altered expression of adhesion-related genes. The enhanced bioprocess properties of this alternative pluripotent state provide a strategy to overcome cell manufacturing limitations of hPSC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1714099115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6016797PMC
June 2018

Trained Memory of Human Uterine NK Cells Enhances Their Function in Subsequent Pregnancies.

Immunity 2018 05;48(5):951-962.e5

The Concern Foundation Laboratories at the Lautenberg Centre for Immunology and Cancer Research, IMRIC, Hebrew University Faculty of Medicine, Jerusalem, Israel. Electronic address:

Natural killer cells (NKs) are abundant in the human decidua, regulating trophoblast invasion and angiogenesis. Several diseases of poor placental development are associated with first pregnancies, so we thus looked to characterize differences in decidual NKs (dNKs) in first versus repeated pregnancies. We discovered a population found in repeated pregnancies, which has a unique transcriptome and epigenetic signature, and is characterized by high expression of the receptors NKG2C and LILRB1. We named these cells Pregnancy Trained decidual NK cells (PTdNKs). PTdNKs have open chromatin around the enhancers of IFNG and VEGFA. Activation of PTdNKs led to increased production and secretion of IFN-γ and VEGFα, with the latter supporting vascular sprouting and tumor growth. The precursors of PTdNKs seem to be found in the endometrium. Because repeated pregnancies are associated with improved placentation, we propose that PTdNKs, which are present primarily in repeated pregnancies, might be involved in proper placentation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2018.03.030DOI Listing
May 2018

Human Brain Organoids on a Chip Reveal the Physics of Folding.

Nat Phys 2018 May 19;14(5):515-522. Epub 2018 Feb 19.

Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel, 7610001.

Human brain wrinkling has been implicated in neurodevelopmental disorders and yet its origins remain unknown. Polymer gel models suggest that wrinkling emerges spontaneously due to compression forces arising during differential swelling, but these ideas have not been tested in a living system. Here, we report the appearance of surface wrinkles during the development and self-organization of human brain organoids in a micro-fabricated compartment that supports imaging over a timescale of weeks. We observe the emergence of convolutions at a critical cell density and maximal nuclear strain, which are indicative of a mechanical instability. We identify two opposing forces contributing to differential growth: cytoskeletal contraction at the organoid core and cell-cycle-dependent nuclear expansion at the organoid perimeter. The wrinkling wavelength exhibits linear scaling with tissue thickness, consistent with balanced bending and stretching energies. Lissencephalic (smooth brain) organoids display reduced convolutions, modified scaling and a reduced elastic modulus. Although the mechanism here does not include the neuronal migration seen in , it models the physics of the folding brain remarkably well. Our on-chip approach offers a means for studying the emergent properties of organoid development, with implications for the embryonic human brain.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41567-018-0046-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947782PMC
May 2018

mA deposition: a boost from TGFβ.

Cell Res 2018 May;28(5):505-506

The Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, 7610001, Israel.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41422-018-0037-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943575PMC
May 2018

RAS Regulates the Transition from Naive to Primed Pluripotent Stem Cells.

Stem Cell Reports 2018 03 15;10(3):1088-1101. Epub 2018 Feb 15.

Department of Genetics and Developmental Biology, The Rappaport Faculty of Medicine and Research Institute, Technion - Israel Institute of Technology, Haifa 31096, Israel. Electronic address:

The transition from naive to primed state of pluripotent stem cells is hallmarked by epithelial-mesenchymal transition, metabolic switch from oxidative phosphorylation to aerobic glycolysis, and changes in the epigenetic landscape. Since these changes are also seen as putative hallmarks of neoplastic cell transformation, we hypothesized that oncogenic pathways may be involved in this process. We report that the activity of RAS is repressed in the naive state of mouse embryonic stem cells (ESCs) and that all three RAS isoforms are significantly activated upon early differentiation induced by LIF withdrawal, embryoid body formation, or transition to the primed state. Forced expression of active RAS and RAS inhibition have shown that RAS regulates glycolysis, CADHERIN expression, and the expression of repressive epigenetic marks in pluripotent stem cells. Altogether, this study indicates that RAS is located at a key junction of early ESC differentiation controlling key processes in priming of naive cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2018.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5918191PMC
March 2018

NKp46 Receptor-Mediated Interferon-γ Production by Natural Killer Cells Increases Fibronectin 1 to Alter Tumor Architecture and Control Metastasis.

Immunity 2018 01 9;48(1):107-119.e4. Epub 2018 Jan 9.

The Lautenberg Center for General and Tumor Immunology, Department of Immunology and Cancer Research, IMRIC, Faculty of Medicine, The Hebrew University Medical School, Jerusalem, Israel. Electronic address:

Natural killer (NK) cells are innate lymphoid cells, and their presence within human tumors correlates with better prognosis. However, the mechanisms by which NK cells control tumors in vivo are unclear. Here, we used reflectance confocal microscopy (RCM) imaging in humans and in mice to visualize tumor architecture in vivo. We demonstrated that signaling via the NK cell receptor NKp46 (human) and Ncr1 (mouse) induced interferon-γ (IFN-γ) secretion from intratumoral NK cells. NKp46- and Ncr1-mediated IFN-γ production led to the increased expression of the extracellular matrix protein fibronectin 1 (FN1) in the tumors, which altered primary tumor architecture and resulted in decreased metastases formation. Injection of IFN-γ into tumor-bearing mice or transgenic overexpression of Ncr1 in NK cells in mice resulted in decreased metastasis formation. Thus, we have defined a mechanism of NK cell-mediated control of metastases in vivo that may help develop NK cell-dependent cancer therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2017.12.007DOI Listing
January 2018

Relevance of iPSC-derived human PGC-like cells at the surface of embryoid bodies to prechemotaxis migrating PGCs.

Proc Natl Acad Sci U S A 2017 11 30;114(46):E9913-E9922. Epub 2017 Oct 30.

Massachusetts General Hospital Center for Cancer Research and Harvard Medical School, Charlestown, MA 02129;

Pluripotent stem cell-derived human primordial germ cell-like cells (hPGCLCs) provide important opportunities to study primordial germ cells (PGCs). We robustly produced CD38 hPGCLCs [∼43% of FACS-sorted embryoid body (EB) cells] from primed-state induced pluripotent stem cells (iPSCs) after a 72-hour transient incubation in the four chemical inhibitors (4i)-naïve reprogramming medium and showed transcriptional consistency of our hPGCLCs with hPGCLCs generated in previous studies using various and distinct protocols. Both CD38 hPGCLCs and CD38 EB cells significantly expressed and , although PRDM1 mRNA in CD38 cells lacked the 3'-UTR harboring miRNA binding sites regulating mRNA stability. Genes up-regulated in hPGCLCs were enriched for cell migration genes, and their promoters were enriched for the binding motifs of TFAP2 (which was identified in promoters of , , and ) and the RREB-1 cell adhesion regulator. In EBs, hPGCLCs were identified exclusively in the outermost surface monolayer as dispersed cells or cell aggregates with strong and specific expression of POU5F1/OCT4 protein. Time-lapse live cell imaging revealed active migration of hPGCLCs on Matrigel. Whereas all hPGCLCs strongly expressed the CXCR4 chemotaxis receptor, its ligand CXCL12/SDF1 was not significantly expressed in the whole EBs. Exposure of hPGCLCs to CXCL12/SDF1 induced cell migration genes and antiapoptosis genes. Thus, our study shows that transcriptionally consistent hPGCLCs can be readily produced from hiPSCs after transition of their pluripotency from the primed state using various methods and that hPGCLCs resemble the early-stage PGCs randomly migrating in the midline region of human embryos before initiation of the CXCL12/SDF1-guided chemotaxis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1707779114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5699045PMC
November 2017

OCT4 impedes cell fate redirection by the melanocyte lineage master regulator MITF in mouse ESCs.

Nat Commun 2017 10 18;8(1):1022. Epub 2017 Oct 18.

Department of Human Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, 69978, Israel.

Ectopic expression of lineage master regulators induces transdifferentiation. Whether cell fate transitions can be induced during various developmental stages has not been systemically examined. Here we discover that amongst different developmental stages, mouse embryonic stem cells (mESCs) are resistant to cell fate conversion induced by the melanocyte lineage master regulator MITF. By generating a transgenic system we exhibit that in mESCs, the pluripotency master regulator Oct4, counteracts pro-differentiation induced by Mitf by physical interference with MITF transcriptional activity. We further demonstrate that mESCs must be released from Oct4-maintained pluripotency prior to ectopically induced differentiation. Moreover, Oct4 induction in various differentiated cells represses their lineage identity in vivo. Alongside, chromatin architecture combined with ChIP-seq analysis suggest that Oct4 competes with various lineage master regulators for binding promoters and enhancers. Our analysis reveals pluripotency and transdifferentiation regulatory principles and could open new opportunities in the field of regenerative medicine.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-017-01122-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647326PMC
October 2017

Increased NK cell immunity in a transgenic mouse model of NKp46 overexpression.

Sci Rep 2017 10 12;7(1):13090. Epub 2017 Oct 12.

The Lautenberg Center for General and Tumor Immunology, Department of Immunology and Cancer Research, IMRIC, Faculty of Medicine, The Hebrew University Medical School, Jerusalem, Israel.

Natural Killer (NK) cells employ activating receptors like the Natural Cytotoxicity Receptors (NCRs: NKp30, NKp44 and NKp46), of which only NKp46 has a mouse orthologue (Ncr1), to eliminate abnormal cells. NKp46/Ncr1 is considered a selective marker for NK cells, although it is also found on a subset of ILCs, where it appears to be without function. The influenza virus hemagglutinin (HA) was the first ligand identified for Ncr1/NKp46 followed by other viral, bacterial and even fungal ligands. NKp46/Ncr1 also recognizes unknown self and tumor ligands. Here we describe the generation of a transgenic mouse where the Ncr1 gene is expressed in the Rosa locus, preceded by a floxed stop sequence allowing Ncr1/NKp46 expression in various tissues upon crossing with Cre transgenic mouse lines. Surprisingly, while several crossings were attempted, Ncr1 overexpression was successful only where cre recombinase expression was dependent on the Ncr1 promoter. Ncr1 overexpression in NK cells increased NK cell immunity in two hallmark Ncr1 related pathologies, influenza virus infection and B16 melanoma. These data suggest that increasing NK cell cytotoxicity by enforced NKp46/Ncr1 expression serves as a potential therapeutic opportunity for the treatment of various pathologies, and in immunotherapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-12998-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5638832PMC
October 2017

A multiplexed screening method for pluripotency.

Stem Cell Res 2017 08 12;23:158-162. Epub 2017 Jul 12.

The Nancy & Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science, Rehovot, Israel. Electronic address:

Measurement of Alkaline Phosphatase (ALP) level is a widely used procedure in clinical and basic research. We present a simple and inexpensive luminescence-based method that allows multiplexed measurement and normalization of intracellular ALP levels in one sample well. The method comprises two commercially available reagents enabling quantification of ALP levels and cell number by two sequential luminescence readouts. Using this method we were able to detect and analyze somatic reprogramming into pluripotent stem cells. The method is highly applicable for High Throughput Screening (HTS) campaigns and analysis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.scr.2017.07.014DOI Listing
August 2017

mA mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover.

Genes Dev 2017 05;31(10):990-1006

Laboratory of Molecular Neuro-Oncology, The Rockefeller University, New York, New York 10065, USA.

Understanding the biologic role of -methyladenosine (mA) RNA modifications in mRNA requires an understanding of when and where in the life of a pre-mRNA transcript the modifications are made. We found that HeLa cell chromatin-associated nascent pre-mRNA (CA-RNA) contains many unspliced introns and mA in exons but very rarely in introns. The mA methylation is essentially completed upon the release of mRNA into the nucleoplasm. Furthermore, the content and location of each mA modification in steady-state cytoplasmic mRNA are largely indistinguishable from those in the newly synthesized CA-RNA or nucleoplasmic mRNA. This result suggests that quantitatively little methylation or demethylation occurs in cytoplasmic mRNA. In addition, only ∼10% of mAs in CA-RNA are within 50 nucleotides of 5' or 3' splice sites, and the vast majority of exons harboring mA in wild-type mouse stem cells is spliced the same in cells lacking the major mA methyltransferase Mettl3. Both HeLa and mouse embryonic stem cell mRNAs harboring mAs have shorter half-lives, and thousands of these mRNAs have increased half-lives (twofold or more) in Mettl3 knockout cells compared with wild type. In summary, mA is added to exons before or soon after exon definition in nascent pre-mRNA, and while mA is not required for most splicing, its addition in the nascent transcript is a determinant of cytoplasmic mRNA stability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/gad.301036.117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495127PMC
May 2017

Transcriptional programs that control expression of the autoimmune regulator gene Aire.

Nat Immunol 2017 02 12;18(2):161-172. Epub 2016 Dec 12.

Department of Immunology, Weizmann Institute of Science, Rehovot, Israel.

Aire is a transcriptional regulator that induces promiscuous expression of thousands of genes encoding tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs). While the target genes of Aire are well characterized, the transcriptional programs that regulate its own expression have remained elusive. Here we comprehensively analyzed both cis-acting and trans-acting regulatory mechanisms and found that the Aire locus was insulated by the global chromatin organizer CTCF and was hypermethylated in cells and tissues that did not express Aire. In mTECs, however, Aire expression was facilitated by concurrent eviction of CTCF, specific demethylation of exon 2 and the proximal promoter, and the coordinated action of several transcription activators, including Irf4, Irf8, Tbx21, Tcf7 and Ctcfl, which acted on mTEC-specific accessible regions in the Aire locus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/ni.3638DOI Listing
February 2017