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1. Variants in SART3 cause a spliceosomopathy characterised by failure of testis development and neuronal defects

Author

Ayers, Katie, Eggers, Stefanie, Rollo, Ben, Smith, Katherine, Davidson, Nadia, Siddall, Nicole, Zhao, Liang, Bowles, Josephine, Weiss, Karin, Zanni, Ginevra, Burglen, Lydie, Ben-Shachar, Shay, Rosensaft, Jenny, Raas-Rothschild, Annick, Jørgensen, Anne, Schittenhelm, Ralf, Huang, Cheng, Robevska, Gorjana, van den Bergen, Jocelyn, Casagranda, Franca, Cyza, Justyna, Pachernegg, Svenja, Wright, David, Bahlo, Melanie, Oshlack, Alicia, O'Brien, Terrence, Kwan, Patrick, Koopman, Peter, Hime, Gary, Girard, Nadine, Hoffmann, Chen, Shilon, Yuval, Zung, Amnon, Bertini, Enrico, Milh, Mathieu, Ben Rhouma, Bochra, Belguith, Neila, Bashamboo, Anu, Mcelreavey, Ken, Banne, Ehud, Weintrob, Naomi, Benzeev, Bruria, Sinclair, Andrew, Murdoch Children's Research Institute (MCRI), University of Melbourne, Victorian Clinical Genetics Services, Monash University [Melbourne], The Walter and Eliza Hall Institute of Medical Research (WEHI), University of Queensland [Brisbane], Technion - Israel Institute of Technology [Haifa], Bambino Gesù Children’s Hospital [Rome, Italy], CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Génétique des Troubles du Neurodéveloppement = Developmental Brain Disorders Laboratory (Equipe Inserm U1163), Imagine - Institut des maladies génétiques (IHU) (Imagine - U1163), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Cité (UPCité), Tel Aviv Sourasky Medical Center [Te Aviv], The Hebrew University Hadassah Medical School, Chaim Sheba Medical Center, Tel Aviv University (TAU), Rigshospitalet [Copenhagen], Copenhagen University Hospital, Monash University [Clayton], Peter Mac Callum Cancer Centre, Service de pédiatrie et neurologie pédiatrique, Université de la Méditerranée - Aix-Marseille 2-Assistance Publique - Hôpitaux de Marseille (APHM)- Hôpital de la Timone [CHU - APHM] (TIMONE), Kaplan Medical Center [Rehovot, Israel], Université de Gabès, Université de Sfax - University of Sfax, Hôpital Charles Nicolle [Tunis], Génétique du Développement humain - Human developmental genetics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Wolfson Medical Center, This study was supported by a National Health and Medical Research Council (NHMRC) programme grant (1074258) awarded to AS, NHMRC project grant (1156942) (K.A.), a Medical Research Future Fund Stem Cells Mission grant (MRF1201781) (K.A., B.N.R. and P.Kw), an Australian Research Council Future Fellowship (FT100100764) to M.B., A NHMRC Investigator Grant (1174040) to D.W., Agence Nationale de la Recherche funding ANR-10-LABX-73 REVIVE, ANR-17-CE14-0038-01 and ANR 20 CE14 0007 to K.M., ANR-19-CE140022 and ANR-19-CE14-0012 to A.B., G.Z. and E.B. are members of the European Reference Network for Rare Neurological Diseases - Project ID No 739510., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-20-CE14-0007,Goldilocks,Analyse intégrée du rôle du facteur de transcription SF-1 / NR5A1 et de ses gènes cibles dépendants du dosage dans la fonction gonadique et les troubles du développement sexuel (DSD)(2020), ANR-19-CE14-0022,SexDiff,Régulation de la détermination du sexe et de la différenciation ovarienne : implications dans les troubles du développement sexuel(2019), and ANR-19-CE14-0012,RNA-SEX,Fonction de l'ARN hélicase dans la détermination du sexe chez les vertébrés et les troubles du développement du sexe chez l'homme (DSD)(2019)

Subjects
MESH: Humans, MESH: RNA-Binding Proteins, MESH: Testis, [SDV]Life Sciences [q-bio], MESH: Gonadal Dysgenesis, MESH: Antigens, Neoplasm, MESH: Induced Pluripotent Stem Cells, MESH: Male, MESH: Intellectual Disability
Abstract

International audience; Squamous cell carcinoma antigen recognized by T cells 3 ( SART3 ) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome ( I ntellectual disability, N eurodevelopmental defects and D evelopmental delay with 46,X Y GON adal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

Published
2023

2. In vitro cellular reprogramming to model gonad development and its disorders

Author

Gonen, Nitzan, Eozenou, Caroline, Mitter, Richard, Elzaiat, Maëva, Stévant, Isabelle, Aviram, Rona, Bernardo, Andreia Sofia, Chervova, Almira, Wankanit, Somboon, Frachon, Emmanuel, Commere, Pierre-Henri, Brailly-Tabard, Sylvie, Valon, Léo, Barrio Cano, Laura, Levayer, Romain, Mazen, Inas, Gobaa, Samy, Smith, James C., McElreavey, Kenneth, Lovell-Badge, Robin, Bashamboo, Anu, Bar-Ilan University [Israël], The Francis Crick Institute [London], Génétique du Développement humain - Human developmental genetics, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Imperial College London, Département de Biologie du Développement et Cellules souches - Department of Developmental and Stem Cell Biology, Institut Pasteur [Paris] (IP), Biomatériaux et Microfluidiques (plateforme) - Biomaterials and Microfluidics (platform), Cytometrie et Biomarqueurs – Cytometry and Biomarkers (UTechS CB), AP-HP Hôpital Bicêtre (Le Kremlin-Bicêtre), Mort cellulaire et homéostasie des épithéliums / Cell death and epithelial homeostasis, National Research Center [Caire, Egypte], This work is funded in part by a research grant (40000767) from the European Society of Pediatric Endocrinology (to A.B.) and by the Agence Nationale de la Recherche (ANR, ANR-10-LABX-73 REVIVE, ANR-17-CE14-0038-01, and ANR 20CE14 0007 to K.M., and ANR-19-CE14-0022 and ANR-19-CE14-0012 to A.B.). N.G., A.S.B., R.M., J.C.S., and R.L.-B. were funded by the Francis Crick Institute. The Francis Crick Institute receives its core funding from Cancer Research UK (CC2116), the UK Medical Research Council (CC2116), and the Wellcome Trust (CC2116). For the purpose of Open Access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. . N.G., I.S., and R.A. are funded by the ERC Starting Grant EnhanceSex (101039928). A.S.B. was also funded by the British Heart Foundation (BHF-FS/12/37/29516) and the Wellcome Trust (210987/Z/18/Z)., ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), ANR-17-CE14-0038,MGonDev,Etude des mécanismes du développement des gonades chez l'homme(2017), ANR-20-CE14-0007,Goldilocks,Analyse intégrée du rôle du facteur de transcription SF-1 / NR5A1 et de ses gènes cibles dépendants du dosage dans la fonction gonadique et les troubles du développement sexuel (DSD)(2020), ANR-19-CE14-0022,SexDiff,Régulation de la détermination du sexe et de la différenciation ovarienne : implications dans les troubles du développement sexuel(2019), and ANR-19-CE14-0012,RNA-SEX,Fonction de l'ARN hélicase dans la détermination du sexe chez les vertébrés et les troubles du développement du sexe chez l'homme (DSD)(2019)

Subjects
Model organisms, Chemical Biology & High Throughput, Multidisciplinary, MESH: Humans, FOS: Clinical medicine, Stem Cells, [SDV]Life Sciences [q-bio], Genome Integrity & Repair, Neurosciences, Gene Expression, MESH: Gonadal Dysgenesis, 46,XY, Tumour Biology, MESH: Induced Pluripotent Stem Cells, MESH: Cellular Reprogramming, MESH: Male, Signalling & Oncogenes, MESH: Gonads, MESH: Animals, Genetics & Genomics, MESH: Mice, MESH: Female, Developmental Biology, Computational & Systems Biology
Abstract

During embryonic development, mutually antagonistic signaling cascades determine gonadal fate toward a testicular or ovarian identity. Errors in this process result in disorders of sex development (DSDs), characterized by discordance between chromosomal, gonadal, and anatomical sex. The absence of an appropriate, accessible in vitro system is a major obstacle in understanding mechanisms of sex-determination/DSDs. Here, we describe protocols for differentiation of mouse and human pluripotent cells toward gonadal progenitors. Transcriptomic analysis reveals that the in vitro–derived murine gonadal cells are equivalent to embryonic day 11.5 in vivo progenitors. Using similar conditions, Sertoli-like cells derived from 46,XY human induced pluripotent stem cells (hiPSCs) exhibit sustained expression of testis-specific genes, secrete anti-Müllerian hormone, migrate, and form tubular structures. Cells derived from 46,XY DSD female hiPSCs, carrying an NR5A1 variant, show aberrant gene expression and absence of tubule formation. CRISPR-Cas9–mediated variant correction rescued the phenotype. This is a robust tool to understand mechanisms of sex determination and model DSDs.

Published
2023

3. SNORD116 and growth hormone therapy impact IGFBP7 in Prader–Willi syndrome

Author

Lisa Cole Burnett, Françoise Conte Auriol, Eric Bieth, Jean Pierre Salles, Juliette Salles, Maithé Tauber, Gwenaelle Diene, Catherine Molinas, Rudolph L. Leibel, Boris V. Skryabin, Isabelle Gennero, Timofey S. Rozhdestvensky, Sanaa Eddiry, Centre d'investigation clinique de Toulouse (CIC 1436), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Pôle Santé publique et médecine publique [CHU Toulouse], Centre Hospitalier Universitaire de Toulouse (CHU Toulouse)-Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Référence du Syndrome de Prader-Willi, Pôle Enfants [CHU Toulouse], Service Psychiatrie et psychologie médicale [CHU Toulouse], Pôle Psychiatrie [CHU Toulouse], Institut Fédératif de Biologie (IFB), Centre Hospitalier Universitaire de Toulouse (CHU Toulouse), University Hospital Münster - Universitaetsklinikum Muenster [Germany] (UKM), University of Illinois [Chicago] (UIC), University of Illinois System, Columbia University [New York], and Pistre, Karine

Subjects
0301 basic medicine, MESH: Prader-Willi Syndrome* / genetics, medicine.medical_specialty, MESH: RNA, Small Nucleolar, congenital, hereditary, and neonatal diseases and abnormalities, IGFBP7, [SDV]Life Sciences [q-bio], Induced Pluripotent Stem Cells, MESH: Neurons, MESH: Induced Pluripotent Stem Cells, Article, MESH: Prader-Willi Syndrome* / drug therapy, 03 medical and health sciences, Mice, 0302 clinical medicine, Neurodevelopmental disorder, Downregulation and upregulation, Internal medicine, medicine, Animals, Humans, RNA, Small Nucleolar, MESH: Animals, MESH: Mice, Genetics (clinical), Neurons, MESH: Humans, [SDV.MHEP] Life Sciences [q-bio]/Human health and pathology, business.industry, Dopaminergic, nutritional and metabolic diseases, medicine.disease, Phenotype, Pathophysiology, nervous system diseases, [SDV] Life Sciences [q-bio], 030104 developmental biology, Endocrinology, MESH: Growth Hormone, Growth Hormone, Knockout mouse, business, Genomic imprinting, Prader-Willi Syndrome, 030217 neurology & neurosurgery, [SDV.MHEP]Life Sciences [q-bio]/Human health and pathology
Abstract

International audience; Purpose: Prader-Willi syndrome (PWS) is a neurodevelopmental disorder with hypothalamic dysfunction due to deficiency of imprinted genes located on the 15q11-q13 chromosome. Among them, the SNORD116 gene appears critical for the expression of the PWS phenotype. We aimed to clarify the role of SNORD116 in cellular and animal models with regard to growth hormone therapy (GHT), the main approved treatment for PWS.Methods: We collected serum and induced pluripotent stem cells (iPSCs) from GH-treated PWS patients to differentiate into dopaminergic neurons, and in parallel used a Snord116 knockout mouse model. We analyzed the expression of factors potentially linked to GH responsiveness.Results: We found elevated levels of circulating IGFBP7 in naive PWS patients, with IGFBP7 levels normalizing under GHT. We found elevated IGFBP7 levels in the brains of Snord116 knockout mice and in iPSC-derived neurons from a SNORD116-deleted PWS patient. High circulating levels of IGFBP7 in PWS patients may result from both increased IGFBP7 expression and decreased IGFBP7 cleavage, by downregulation of the proconvertase PC1.Conclusion: SNORD116 deletion affects IGFBP7 levels, while IGFBP7 decreases under GHT in PWS patients. Modulation of the IGFBP7 level, which interacts with IGF1, has implications in the pathophysiology and management of PWS under GHT.

Published
2021

4. Generation of two isogenic induced pluripotent stem cell lines from a 4-month-old severe nemaline myopathy patient with a heterozygous dominant c.553C > A (p.Arg183Ser) variant in the ACTA1 gene

Author

Kristen J. Nowak, Edoardo Malfatti, Thierry Larmonier, Gianina Ravenscroft, Carolin K. Scriba, Safaa Saker, Norma B. Romero, Rhonda L. Taylor, Joshua S. Clayton, Nigel G. Laing, The University of Western Australia (UWA), Institut de Myologie, Centre National de la Recherche Scientifique (CNRS)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Association française contre les myopathies (AFM-Téléthon)-Sorbonne Université (SU), Centre de recherche en Myologie – U974 SU-INSERM, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Centre d'Investigation Clinique Henri Mondor (CIC Henri Mondor), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Henri Mondor-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris-Est Créteil Val-de-Marne - Paris 12 (UPEC UP12), Généthon, This work was supported by funding from an AFM Telethon Trampoline Grant (REF-21816) to Kristen Nowak, and A Foundation Building Strength Research Grant (ID-A3TR22, PI Nigel Laing). We also gratefully acknowledge funding from the Australian National Health and Medical Research Council, including a Principal Research Fellowship (APP1117510) to Nigel Laing and a Career Development Fellowship (APP1122952) to Gianina Ravenscroft., Gestionnaire, Hal Sorbonne Université, Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Association française contre les myopathies (AFM-Téléthon)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS), Université de Versailles Saint-Quentin-en-Yvelines - UFR Sciences de la santé Simone Veil (UVSQ Santé), Université de Versailles Saint-Quentin-en-Yvelines (UVSQ), and Centre de Recherche en Myologie

Subjects
0301 basic medicine, MESH: Mutation, QH301-705.5, MESH: Female, [SDV]Life Sciences [q-bio], Induced Pluripotent Stem Cells, Biology, medicine.disease_cause, Myopathies, Nemaline, MESH: Infant, 03 medical and health sciences, 0302 clinical medicine, Nemaline myopathy, medicine, Humans, Biology (General), Induced pluripotent stem cell, Nemaline bodies, Muscle, Skeletal, Gene, Mutation, Lymphoblast, MESH: Actins / genetics, MESH: Induced Pluripotent Stem Cells, Skeletal muscle, Infant, Cell Biology, General Medicine, medicine.disease, Congenital myopathy, Molecular biology, Actins, 3. Good health, [SDV] Life Sciences [q-bio], MESH: Humans, 030104 developmental biology, medicine.anatomical_structure, MESH: Muscle, Skeletal, MESH: Myopathies, Nemaline* / genetics, Female, 030217 neurology & neurosurgery, Developmental Biology
Abstract

International audience; AbstractNemaline myopathy (NM) is a congenital myopathy typically characterized by skeletal muscle weakness and the presence of abnormal thread- or rod-like structures (nemaline bodies) in myofibres. Pathogenic variants in the skeletal muscle alpha actin gene, ACTA1, cause approximately 25% of all NM cases. We generated two induced pluripotent stem cell lines from lymphoblastoid cells of a 4-month-old female with severe NM harbouring a dominant variant in ACTA1 (c.553C > A). The isogenic lines displayed characteristic iPSC morphology, expressed pluripotency markers, differentiated into cells of all three germ layers, and possessed normal karyotypes. These lines could be useful models of human ACTA1 disease.

Published
2021

5. iPSC reprogramming of fibroblasts from a patient with a Rothmund-Thomson syndrome RTS

Author

Lydiane Pichard, Alice Goldenberg, Franck Pellestor, Xavier Balguerie, Vincent Gatinois, Jean-Marc Lemaitre, Romain Desprat, Fabienne Becker, Cellules Souches, Plasticité Cellulaire, Médecine Régénératrice et Immunothérapies (IRMB), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), CHU Montpellier, Département de génétique [CHU Rouen] (Centre Normandie de Génomique et de Médecine Personnalisée), CHU Rouen, Normandie Université (NU)-Normandie Université (NU), Normandie Université (NU), Hôpital Lapeyronie [Montpellier] (CHU), and Salvy-Córdoba, Nathalie

Subjects
0301 basic medicine, MESH: Mutation, Induced Pluripotent Stem Cells, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Induced Pluripotent Stem Cells, MESH: Rothmund-Thomson Syndrome, Biology, 03 medical and health sciences, 0302 clinical medicine, Cataracts, medicine, Humans, Induced pluripotent stem cell, lcsh:QH301-705.5, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Gene, Rothmund–Thomson syndrome, MESH: Humans, musculoskeletal, neural, and ocular physiology, Rothmund-Thomson Syndrome, Helicase, Cancer, MESH: Skin Abnormalities, Cell Biology, General Medicine, [SDV.MHEP.DERM] Life Sciences [q-bio]/Human health and pathology/Dermatology, Fibroblasts, medicine.disease, 030104 developmental biology, lcsh:Biology (General), MESH: Fibroblasts, Cell culture, Mutation, biology.protein, Cancer research, Skin Abnormalities, sense organs, Reprogramming, [SDV.MHEP.DERM]Life Sciences [q-bio]/Human health and pathology/Dermatology, 030217 neurology & neurosurgery, Developmental Biology
Abstract

International audience; Rothmund-Thomson Syndrome (RTS) is a rare autosomal recessive disease that manifests several clinical features of accelerated aging. These findings include atrophic skin and pigment changes, alopecia, osteopenia, cataracts, and an increased incidence of cancer for patients. Mutations in RECQL4 gene are responsible for cases of RTS. RECQL4 belongs to the RECQ DNA helicase family which has been shown to participate in many aspects of DNA metabolism. To be able to study the cellular defects related to the pathology, we derived an induced pluripotent cell line from RTS patient fibroblasts, with the ability to re-differentiate into the three embryonic germ layers.

Published
2019

6. Human NPCs can degrade α–syn fibrils and transfer them preferentially in a cell contact-dependent manner possibly through TNT-like structures

Author

Masato Hasegawa, Clara Grudina, Takashi Nonaka, Rebecca Matsas, Chiara Zurzolo, Georgia Kouroupi, Trafic membranaire et Pathogénèse, Institut Pasteur [Paris], Institut Pasteur Hellénique, Réseau International des Instituts Pasteur (RIIP), Dementia Research Project, Tokyo Metropolitan Institute of Medical Science (TMIMS), This work was supported by the Programme Transversaux De Recherche - PTR (523) from Institut Pasteur (R.M. and C.Z), Agence Nationale de la Recherche [ANR-16CE-0019-01] (C.Z), Fondation pour la Recherche Médicale [FRM-2016-DEQ20160334896] (C.Z), France Alzheimer [AAP SM 2017#1674] (C.Z), LECMA-VAINCRE ALZHEIMER [2016/FR-16020] (C.Z), Scientific Research on Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) [JP14533254] [JP18dm0207019] from AMED (M.H.), JST CREST (JP18071300) (M.H.)., We are thankful to Dr. Michael Henderson for the proofread of this manuscript, all lab members of CZ group for all suggestions and technical supports in this project., ANR-16-CE16-0019,Neurotunn,Role des nanotubes membranaires dans la propagation d'agrégats protéiques impliqués dans les maladie neurodégénératives(2016), Grudina, C., Kouroupi, G., Nonaka, T., Hasegawa, M., Matsas, R., Zurzolo, C., and Institut Pasteur [Paris] (IP)

Subjects
0301 basic medicine, MESH: Neural Stem Cells, Parkinson's disease, Human neuronal precursors, [SDV]Life Sciences [q-bio], Cell Communication, Degeneration (medical), Induced Pluripotent Stem Cell, chemistry.chemical_compound, 0302 clinical medicine, Neural Stem Cells, MESH: Dopaminergic Neurons, Neural Stem Cell, TNT-like structure, Induced pluripotent stem cell, Endocytosi, Dopaminergic, Lysosome, Endocytosis, Cell biology, Neurology, MESH: Endocytosis, Dopaminergic Neuron, Human, Induced Pluripotent Stem Cells, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, MESH: Induced Pluripotent Stem Cells, Fibril, lcsh:RC321-571, Midbrain, Alpha-synuclein, 03 medical and health sciences, MESH: Cell Communication, MESH: alpha-Synuclein, alpha-Synuclein, medicine, Humans, lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry, MESH: Humans, Dopaminergic Neurons, medicine.disease, Transplantation, TNT-like structures, Human neuronal precursor, 030104 developmental biology, chemistry, nervous system, Lysosomes, 030217 neurology & neurosurgery, MESH: Lysosomes
Abstract

International audience; Parkinson's disease (PD) is the second most common neurodegenerative disorder whereby loss of midbrain dopaminergic neurons results in motor dysfunction. Transplantation of human induced pluripotent stem cells (iPSCs) into the brain of patients affected by PD is one of the therapeutic approaches that has gained interest to compensate for the degeneration of neurons and improve disease symptoms. However, only a part of transplanted cells can differentiate into mature neurons while the majority remains in undifferentiated state. Here we investigated whether human neuronal precursor cells (hNPCs) derived from iPSCs have an active role in α-synuclein (α-syn) pathology. Our findings demonstrate that α-syn fibrils are taken up by hNPCs and are preferentially localized in lysosomes where they can be degraded. However, α-syn fibrils are also transferred between hNPCs in a cell-to-cell contact dependent manner, and are found in tunneling nanotube (TNT)-like structures. Thus, NPCs can have a dual role in the progression of α-syn pathology, which should be considered in human transplants.

Published
2019

7. Establishment of a collection of human pluripotent stem cell lines (iPSC) from mesenchymal stem cells (MSC) from three healthy elderly donors

Author

Fabienne Becker, P. Pastoureau, Christian Jorgensen, Danièle Noël, Lydiane Pichard, Frédéric De Ceuninck, Jean-Marc Lemaitre, Romain Desprat, Jean-Marc Brondello, Cellules Souches, Plasticité Cellulaire, Médecine Régénératrice et Immunothérapies (IRMB), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Institut de Recherches SERVIER (IRS), and Retiveau, Nolwenn

Subjects
MESH: Cell Differentiation, Pluripotent Stem Cells, 0301 basic medicine, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, QH301-705.5, Cellular differentiation, Induced Pluripotent Stem Cells, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Disease, MESH: Induced Pluripotent Stem Cells, Biology, MESH: Cellular Reprogramming, Cell Line, 03 medical and health sciences, 0302 clinical medicine, medicine, Humans, Epigenetics, Biology (General), Induced pluripotent stem cell, [SDV.BC] Life Sciences [q-bio]/Cellular Biology, Aged, MESH: Aged, MESH: Humans, Cartilage, Mesenchymal stem cell, Cell Differentiation, Mesenchymal Stem Cells, Cell Biology, General Medicine, Cellular Reprogramming, MESH: Cell Line, [SDV.BIO] Life Sciences [q-bio]/Biotechnology, MESH: Mesenchymal Stem Cells, 030104 developmental biology, medicine.anatomical_structure, Cell culture, Cancer research, MESH: Pluripotent Stem Cells, Reprogramming, 030217 neurology & neurosurgery, Developmental Biology
Abstract

International audience; The study of molecular mechanism driving osteoarticular diseases like osteoarthritis or osteoporosis is impaired by the low accessibility to mesenchymal stem cells (MSC) from healthy donors (HD) for differential multi-omics analysis. Advances in cell reprogramming have, however, provided both a new source of human cells for laboratory research and a strategy to erase epigenetic marks involved in cell identity and the development of diseases. To unravel the pathological signatures on the MSC at the origin of cellular drifts during the formation of bone and cartilage, we previously developed iPSC from MSC of osteoarthritis donors. Here we present the derivation of three iPSCs from healthy age matched donors to model the disease and further identify (epi)genomic signatures of the pathology.

Published
2021

8. BMP4 patterns Smad activity and generates stereotyped cell fate organisation in spinal organoids

Author

Nathalie Duval, Célia Vaslin, Xavier Baudin, Vanessa Ribes, Youcef El-Mokhtar Frarma, Stéphane Nedelec, Vincent Contremoulins, Tiago Costa Barata, Institut Jacques Monod (IJM (UMR_7592)), Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Département de Biologie du Développement et Cellules souches - Department of Developmental and Stem Cell Biology, Institut Pasteur [Paris] (IP), Bases Génétiques, Moléculaires et Cellulaires du Développement (BGMCD), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Institut du Fer à Moulin (IFM - Inserm U1270 - SU), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), Stem cells in neurodevelopment (SCN), Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU), ImagoSeine, Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), S.N. and V.C.R. are Institut National de la Santé et de la Recherche Médicale researchers, N.D. is employed by the Institut Pasteur. Work in the lab of V.R. is supported by a Centre National pour la Recherche Scientifique/Institut National de la Santé et de la Recherche Médicale ATIP-AVENIR program, as well as by a Ligue Nationale Contre le Cancer grant (PREAC2016.LCC). Studies in the lab of S.N. are funded by an Institut National de la Santé et de la Recherche Médicale ATIP-AVENIR program co-sponsored by the Association Française contre les Myopathies (AFM-telethon) and a chair of excellence of the Laboratoire d'Excellence de Biologie pour la Psychiatrie (Bio-Psy) (11-LABX-0035)., ANR-11-LABX-0035,BIOPSY,Laboratoire de Psychiatrie Biologique(2011), Université de Paris (UP)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], Duval, Nathalie, Laboratoire de Psychiatrie Biologique - - BIOPSY2011 - ANR-11-LABX-0035 - LABX - VALID, and Institut du Fer à Moulin (UMR-S 1270)

Subjects
Organoid, MESH: Signal Transduction, [SDV]Life Sciences [q-bio], MESH: Bone Morphogenetic Protein 4, MESH: Neurons, Smad Proteins, SMAD, Bone Morphogenetic Protein 4, MESH: Organoids, Pluripotent stem cells differentiation, Mice, 0302 clinical medicine, MESH: Gene Expression Regulation, Developmental, MESH: Animals, Induced pluripotent stem cell, Cells, Cultured, Neurons, 0303 health sciences, Neural crest, Gene Expression Regulation, Developmental, Cell Differentiation, Relay and associating spinal dorsal interneurons, MESH: Interneurons, Cell biology, [SDV] Life Sciences [q-bio], Organoids, Patterning, medicine.anatomical_structure, Neural Crest, embryonic structures, MESH: Spine, Signal Transduction, MESH: Cells, Cultured, Self-organization, MESH: Cell Differentiation, Cell type, Neural Tube, animal structures, Induced Pluripotent Stem Cells, Cell fate determination, Biology, MESH: Induced Pluripotent Stem Cells, Bone morphogenetic protein, MESH: Neural Tube, 03 medical and health sciences, Interneurons, Bone morphogenetic proteins, medicine, Animals, Humans, Cell Lineage, Molecular Biology, MESH: Mice, 030304 developmental biology, MESH: Humans, Neural tube, MESH: Embryo, Mammalian, MESH: Smad Proteins, MESH: Cell Lineage, Embryo, Mammalian, MESH: Neural Crest, In vitro, Spine, 030217 neurology & neurosurgery, Developmental Biology
Abstract

Bone Morphogenetic Proteins (BMP) are secreted regulators of cell fate in several developing tissues. In the embryonic spinal cord, they control the emergence of the neural crest, roof plate and distinct subsets of dorsal interneurons. Although a gradient of BMP activity has been proposed to determine cell type identity invivo, whether this is sufficient for pattern formation invitrois unclear. Here, we demonstrate that exposure to BMP4 initiates distinct spatial dynamics of BMP signalling within the self-emerging epithelia of both mouse and human pluripotent stem cells derived spinal organoids. The pattern of BMP signalling results in the stereotyped spatial arrangement of dorsal neural tube cell types and concentration, timing and duration of BMP4 exposure modulate these patterns. Moreover, differences in the duration of competence time-windows between mouse and human account for the species specific tempo of neural differentiation. Together the study describes efficient methods to generate patterned subsets of dorsal interneurons in spinal organoids and supports the conclusion that graded BMP activity orchestrates the spatial organization of the dorsal neural tube cellular diversity in mouse and human.

Published
2019

9. Mutations in ACTL6B Cause Neurodevelopmental Deficits and Epilepsy and Lead to Loss of Dendrites in Human Neurons

Author

Jacques L. Michaud, Jessica Sebastian, Hanrong Wu, Dick Lindhout, Karla Manzano-Vargas, Nuwan C. Hettige, Ingrid M. Wentzensen, Huashan Peng, Amy Crunk, Heika Silviera, Malvin Jefri, Henry Houlden, Fadi F. Hamdan, Zahia Aouabed, Stephanie Efthymiou, Xin Zhang, Renske Oegema, Arnaud Tanti, Jerry Vockley, Gustavo Turecki, Julien van Gils, Amber G. Begtrup, Christina Nassif, Gunnar Houge, Naomichi Matsumoto, Thomas Bourgeron, Jean-François Théroux, Emily Fassi, Noriko Miyake, Robert D. Nicholls, Jose E. Martinez, Kristin Herman, Lilit Antonyan, Philippe M. Campeau, Margaret G. Au, Dominic Nelson, Vincenzo Salpietro, Scott C. Bell, Pierre Priam, Joshua L. Deignan, Gregory M. Cooper, Rune Østern, Han G. Brunner, Dagmar Huhle, Amy Goldstein, John M. Graham, Christine Coubes, Koen L.I. van Gassen, Tabib Dabir, Maria Hafström, Simon Gravel, Sophie Ehresmann, Elsa Rossignol, Ilaria Kolobova, Walla Al-Hertani, Julie A. Lessard, Lionel Carmant, Sonja Martin, Richard Delorme, Carl Ernst, Jolien S. Klein Wassink-Ruiter, Naguib Mechawar, Yoshio Makita, Candice R. Finnila, Rami Abou Jamra, Anne Lortie, Justine Rousseau, Sarju G. Mehta, Lina Ghaloul-Gonzalez, MUMC+: DA Klinische Genetica (5), Klinische Genetica, RS: GROW - R4 - Reproductive and Perinatal Medicine, McGill University = Université McGill [Montréal, Canada], Université de Montréal (UdeM), University of Pittsburgh (PITT), Pennsylvania Commonwealth System of Higher Education (PCSHE), GeneDx [Gaithersburg, MD, USA], University of California [Davis] (UC Davis), University of California, David Geffen School of Medicine [Los Angeles], University of California [Los Angeles] (UCLA), University of California-University of California, University of Calgary, UCL, Institute of Neurology [London], Yokohama City University (YCU), Asahikawa Medical College, Trondheim University, Haukeland University Hospital, University of Bergen (UiB), Washington University School of Medicine in St. Louis, Washington University in Saint Louis (WUSTL), University of Groningen [Groningen], University Medical Center Groningen [Groningen] (UMCG), Children's Hospital of Pittsburgh of UPMC [Etats-Unis], Belfast City Hospital, Génétique humaine et fonctions cognitives - Human Genetics and Cognitive Functions (GHFC (UMR_3571 / U-Pasteur_1)), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Child and Adolescent Psychiatry Department [AP- HP Hôpital Robert Debré], AP-HP Hôpital universitaire Robert-Debré [Paris], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), HudsonAlpha Institute for Biotechnology [Huntsville, AL], Children’s Rehabilitation Service [Mobile, AL] (CRS), Children's Rehabilitation Service [Montgomery, AL] (CRS), University Medical Center [Utrecht], Cambridge University Hospitals - NHS (CUH), University of Cambridge [UK] (CAM), University Hospital Leipzig, Donders Institute for Brain, Cognition and Behaviour, Radboud university [Nijmegen], Maastricht University Medical Centre (MUMC), Maastricht University [Maastricht], Cedars-Sinai Medical Center, CHU Montpellier, Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), We acknowledge funding by FRQS doctoral (S.B.), Indonesian Endowment Fund for Education PhD award (M.J.), CONACYT (Mexico) and MITACS (K.M.V.), Genome Canada and Génome Québec (J.L.M. and E.R.), Canada Research Chairs program (G.T. and C.E.), AMED, MEXT, JST, MHLW, and Takeda Science Foundation (N. Matsumoto), and CIHR grant (C.E. and P.M.C.)., University of California (UC), University of California (UC)-University of California (UC), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Radboud University [Nijmegen], and Faculteit Medische Wetenschappen/UMCG

Subjects
0301 basic medicine, Male, PROTEIN EXPRESSION, Chromosomal Proteins, Non-Histone, Mutant, MESH: Neurons, 0302 clinical medicine, SYNAPTIC PLASTICITY, MESH: Child, Genetics(clinical), Global developmental delay, TRANSCRIPTION, Child, Genetics (clinical), ACTL6B, genetic engineering, intellectual disability, neurodevelopment, seizure, stem cells, Genetics, Neurons, SWI/SNF COMPLEX, MESH: Infant, Hypotonia, Neural stem cell, Chromatin, DNA-Binding Proteins, medicine.anatomical_structure, MESH: Young Adult, MESH: Epilepsy, Child, Preschool, Female, medicine.symptom, Adult, MESH: Mutation, DISORDERS, Induced Pluripotent Stem Cells, Biology, MESH: Induced Pluripotent Stem Cells, MESH: Actins, MESH: Dendrites, Chromatin remodeling, Article, MESH: Chromatin, 03 medical and health sciences, Young Adult, All institutes and research themes of the Radboud University Medical Center, MESH: Chromosomal Proteins, Non-Histone, medicine, Journal Article, Humans, CHROMATIN REMODELING COMPLEX, Progenitor cell, Loss function, MESH: Neurodevelopmental Disorders, MESH: Humans, Epilepsy, Neurodevelopmental disorders Donders Center for Medical Neuroscience [Radboudumc 7], [SCCO.NEUR]Cognitive science/Neuroscience, MESH: Child, Preschool, COFFIN-SIRIS SYNDROME, MEMORY, Infant, MESH: Adult, Dendrites, GENE, MESH: Male, Actins, 030104 developmental biology, Neurodevelopmental Disorders, Mutation, OLIGODENDROCYTE, Neuron, MESH: Female, 030217 neurology & neurosurgery, MESH: DNA-Binding Proteins
Abstract

International audience; We identified individuals with variations in ACTL6B, a component of the chromatin remodeling machinery including the BAF complex. Ten individuals harbored bi-allelic mutations and presented with global developmental delay, epileptic encephalopathy, and spasticity, and ten individuals with de novo heterozygous mutations displayed intellectual disability, ambulation deficits, severe language impairment, hypotonia, Rett-like stereotypies, and minor facial dysmorphisms (wide mouth, diastema, bulbous nose). Nine of these ten unrelated individuals had the identical de novo c.1027G>A (p.Gly343Arg) mutation. Human-derived neurons were generated that recaptured ACTL6B expression patterns in development from progenitor cell to post-mitotic neuron, validating the use of this model. Engineered knock-out of ACTL6B in wild-type human neurons resulted in profound deficits in dendrite development, a result recapitulated in two individuals with different bi-allelic mutations, and reversed on clonal genetic repair or exogenous expression of ACTL6B. Whole-transcriptome analyses and whole-genomic profiling of the BAF complex in wild-type and bi-allelic mutant ACTL6B neural progenitor cells and neurons revealed increased genomic binding of the BAF complex in ACTL6B mutants, with corresponding transcriptional changes in several genes including TPPP and FSCN1, suggesting that altered regulation of some cytoskeletal genes contribute to altered dendrite development. Assessment of bi-alleic and heterozygous ACTL6B mutations on an ACTL6B knock-out human background demonstrated that bi-allelic mutations mimic engineered deletion deficits while heterozygous mutations do not, suggesting that the former are loss of function and the latter are gain of function. These results reveal a role for ACTL6B in neurodevelopment and implicate another component of chromatin remodeling machinery in brain disease.

Published
2019

10. High N-glycan multiplicity is critical for neuronal adhesion and sensitizes the developing cerebellum to N-glycosylation defect

Author

Medina-Cano, Daniel, Ucuncu, Ekin, Nguyen, Lam Son, Nicouleau, Michael, Lipecka, Joanna, Bizot, Jean-Charles, Thiel, Christian, Foulquier, François, Lefort, Nathalie, Faivre-Sarrailh, Catherine, Colleaux, Laurence, Guerrera, Ida Chiara, Cantagrel, Vincent, Imagine - Institut des maladies génétiques (IMAGINE - U1163), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de chimie et procédés pour l'énergie, l'environnement et la santé (ICPEES), Université de Strasbourg (UNISTRA)-Matériaux et nanosciences d'Alsace (FMNGE), Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut de Chimie du CNRS (INC)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), CHU Trousseau [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), JRC Institute for Energy and Transport (IET), European Commission - Joint Research Centre [Petten], Institut de Neurobiologie de la Méditerranée [Aix-Marseille Université] (INMED - INSERM U1249), Institut National de la Santé et de la Recherche Médicale (INSERM)-Aix Marseille Université (AMU), Structure Fédérative de Recherche Necker (SFR Necker - UMS 3633 / US24), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Université de Strasbourg (UNISTRA)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et nanosciences d'Alsace, Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre National de la Recherche Scientifique (CNRS)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Assistance publique - Hôpitaux de Paris (AP-HP) (APHP), PRA, Fanny, Mécanismes développementaux des anomalies structurelles cérébelleuses - - SCD-Mec2016 - ANR-16-CE12-0005 - AAPG2016 - VALID, ERA-NET rare disease research implementing IRDiRC objectives - E-Rare-3 - - H20202014-12-01 - 2019-11-30 - 643578 - VALID, Institut de psychiatrie et neurosciences (U894 / UMS 1266), Key-Obs, ANR-16-CE12-0005,SCD-Mec,Mécanismes développementaux des anomalies structurelles cérébelleuses(2016), European Project: 643578,H2020,H2020-HCO-2014,E-Rare-3(2014), Université de Strasbourg (UNISTRA)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Matériaux et Nanosciences Grand-Est (MNGE), Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS)-Université de Strasbourg (UNISTRA)-Université de Haute-Alsace (UHA) Mulhouse - Colmar (Université de Haute-Alsace (UHA))-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Unité de Glycobiologie Structurale et Fonctionnelle - UMR 8576 (UGSF), Institut National de la Recherche Agronomique (INRA)-Centre National de la Recherche Scientifique (CNRS)-Université de Lille, Centre de recherche en neurobiologie - neurophysiologie de Marseille (CRN2M), Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université Sorbonne Paris Cité (USPC), and Centre de Psychiatrie et Neurosciences (U894)

Subjects
Glycosylation, MESH: Immunoglobulins, [SDV]Life Sciences [q-bio], MESH: Neurons, N-glycosylation, MESH: Unfolded Protein Response, MESH: Mice, Knockout, neuroscience, Purkinje Cells, MESH: Axon Guidance, Neural Pathways, cell biology, MESH: Animals, Biology (General), Mice, Knockout, Neurons, neuronal migration, MESH: Proteomics, Srd5a3, Cell Differentiation, MESH: Glycosylation, MESH: Motor Activity, Axon Guidance, MESH: Reproducibility of Results, MESH: Cell Adhesion Molecules, Medicine, lipids (amino acids, peptides, and proteins), [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Membrane Proteins, Research Article, MESH: Cell Differentiation, MESH: Mutation, animal structures, cerebellum, QH301-705.5, Science, Induced Pluripotent Stem Cells, Immunoglobulins, MESH: Cytoplasmic Granules, Motor Activity, MESH: Induced Pluripotent Stem Cells, Cytoplasmic Granules, MESH: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase, MESH: Cell Adhesion, MESH: Purkinje Cells, proteomics, 3-Oxo-5-alpha-Steroid 4-Dehydrogenase, Polysaccharides, Animals, human, [SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], mouse, MESH: Neural Pathways, Cell Membrane, Membrane Proteins, Reproducibility of Results, cell adhesion, MESH: Cerebellum, carbohydrates (lipids), MESH: Polysaccharides, [SDV.GEN.GH]Life Sciences [q-bio]/Genetics/Human genetics, MESH: Gene Deletion, Protein N-glycosylation, Mutation, Unfolded Protein Response, Cell Adhesion Molecules, Gene Deletion, MESH: Cell Membrane, congenital disorders of glycosylation
Abstract

International audience; Proper brain development relies highly on protein N-glycosylation to sustain neuronal migration, axon guidance and synaptic physiology. Impairing the N-glycosylation pathway at early steps produces broad neurological symptoms identified in congenital disorders of glycosylation. However, little is known about the molecular mechanisms underlying these defects. We generated a cerebellum specific knockout mouse for Srd5a3, a gene involved in the initiation of N-glycosylation. In addition to motor coordination defects and abnormal granule cell development, Srd5a3 deletion causes mild N-glycosylation impairment without significantly altering ER homeostasis. Using proteomic approaches, we identified that Srd5a3 loss affects a subset of glycoproteins with high N-glycans multiplicity per protein and decreased protein abundance or N-glycosylation level. As IgSF-CAM adhesion proteins are critical for neuron adhesion and highly N-glycosylated, we observed impaired IgSF-CAM-mediated neurite outgrowth and axon guidance in Srd5a3 mutant cerebellum. Our results link high N-glycan multiplicity to fine-tuned neural cell adhesion during mammalian brain development.

Published
2018

11. Modeling amyotrophic lateral sclerosis in pure human iPSc-derived motor neurons isolated by a novel FACS double selection technique

Author

Dorothée Buttigieg, Stéphane Blanchard, Diana Toli, Delphine Bohl, Georg Haase, Gilbert Baillat, Sarah Bellouze, Thomas Lemonnier, Boris Lamotte d'Incamps, Biothérapies pour les Maladies Neurodégénératives, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Institut de Neurosciences de la Timone (INT), Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), Centre de neurophysique, physiologie, pathologie (UMR 8119), Université Paris Descartes - Paris 5 (UPD5)-Centre National de la Recherche Scientifique (CNRS), This work was supported by grants from Agence Nationale pour la Recherche (Modeling ALS with IPS-derived motor neurons, IPSOALS, ANR-08-NEUR-005-02) in the frame of the ERANET-NEURON program. Work in the laboratory of Georg Haase is further financed by CNRS, Aix-Marseille University, Fédération pour la Recherche sur le Cerveau (FRC/Rotary Grant 'Protein aggregates in motoneuron subpopulations: the therapeutic target?'), Association Française contre les Myopathies (AFM grant, 'Motoneuron subpopulations in development and disease'). Work in the laboratory of D. Bohl is supported by INSERM, Institut Pasteur, Agence Nationale de la Recherche (Laboratoire d’Excellence Revive, Investissement d’Avenir, ANR-10-LABX-73), Association Française contre les Myopathies (AFM grant 'Characterisation of pathological defects in motor neurons derived from patients with amyotrophic lateral sclerosis'), Association pour la Recherche sur la Sclerose Laterale Amyotrophique et autres Maladies du Motoneurone (ARS grant 'Human motor neurons genetically reprogrammed from patient fibroblasts: a new approach to analyse ALS physiopathology'), and the Thierry Latran foundation. D. Toli was the recipient of an A.S. Onassis Foundation Scholarship., ANR-09-NEUR-0005,MODDIFSYN(2009), ANR-10-LABX-0073,REVIVE,Stem Cells in Regenerative Biology and Medicine(2010), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), and Aix Marseille Université (AMU)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Cell, MESH: Flow Cytometry, MESH: Nerve Degeneration, Superoxide Dismutase-1, Genes, Reporter, MESH: Child, MESH: Nerve Tissue Proteins, Amyotrophic lateral sclerosis (ALS), Axon, Amyotrophic lateral sclerosis, Child, Induced pluripotent stem cell, Cells, Cultured, MESH: Amyotrophic Lateral Sclerosis, MESH: Superoxide Dismutase, MESH: Lentivirus, MESH: Superoxide Dismutase-1, Motor Neurons, Myogenesis, Flow Cytometry, medicine.anatomical_structure, MESH: Cell Survival, Neurology, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], MESH: Motor Neurons, MESH: Cells, Cultured, Adult, Induced pluripotent stem cells (iPSc), Cell death, MESH: Axons, Cell type, Programmed cell death, MESH: Mutation, Axon degeneration, Cell Survival, Induced Pluripotent Stem Cells, Nerve Tissue Proteins, Receptors, Nerve Growth Factor, MESH: Induced Pluripotent Stem Cells, Biology, lcsh:RC321-571, MESH: Coculture Techniques, p75NTR, MESH: Receptors, Nerve Growth Factor, medicine, Humans, Motor neuron disease, lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry, Fluorescent-activated cell sorting (FACS), MESH: Humans, Superoxide Dismutase, Amyotrophic Lateral Sclerosis, Lentivirus, MESH: Genes, Reporter, MESH: Adult, Motor neuron, medicine.disease, Axons, Coculture Techniques, MESH: Astrocytes, HB9, nervous system, Astrocytes, Mutation, Nerve Degeneration, Superoxide dismutase 1 (SOD1), Neuroscience
Abstract

International audience; Amyotrophic lateral sclerosis (ALS) is a severe and incurable neurodegenerative disease. Human motor neurons generated from induced pluripotent stem cells (iPSc) offer new perspectives for disease modeling and drug testing in ALS. In standard iPSc-derived cultures, however, the two major phenotypic alterations of ALS--degeneration of motor neuron cell bodies and axons--are often obscured by cell body clustering, extensive axon criss-crossing and presence of unwanted cell types. Here, we succeeded in isolating 100% pure and standardized human motor neurons by a novel FACS double selection based on a p75(NTR) surface epitope and an HB9::RFP lentivirus reporter. The p75(NTR)/HB9::RFP motor neurons survive and grow well without forming clusters or entangled axons, are electrically excitable, contain ALS-relevant motor neuron subtypes and form functional connections with co-cultured myotubes. Importantly, they undergo rapid and massive cell death and axon degeneration in response to mutant SOD1 astrocytes. These data demonstrate the potential of FACS-isolated human iPSc-derived motor neurons for improved disease modeling and drug testing in ALS and related motor neuron diseases.

Published
2015

12. Therapeutic effects of proteoliposomes on X-linked chronic granulomatous disease: proof of concept using macrophages differentiated from patient-specific induced pluripotent stem cells

Author

Sandra Cortès, Marie José Stasia, Jean-Luc Lenormand, Julie Brault, Guillaume Vaganay, Aline Le Roy, Université Grenoble Alpes - UFR Pharmacie (UGA UFRP), Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Synthelis, Institut de biologie structurale (IBS - UMR 5075 ), Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Centre National de la Recherche Scientifique (CNRS), Techniques de l'Ingénierie Médicale et de la Complexité - Informatique, Mathématiques et Applications, Grenoble - UMR 5525 (TIMC-IMAG), Institut polytechnique de Grenoble - Grenoble Institute of Technology (Grenoble INP )-VetAgro Sup - Institut national d'enseignement supérieur et de recherche en alimentation, santé animale, sciences agronomiques et de l'environnement (VAS)-Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019]), Centre National de la Recherche Scientifique (CNRS)-Université Grenoble Alpes [2016-2019] (UGA [2016-2019])-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), and Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)

Subjects
0301 basic medicine, Pharmaceutical Science, chronic granulomatous disease, Granulomatous Disease, Chronic, law.invention, Cell membrane, chemistry.chemical_compound, Chronic granulomatous disease, MESH: Granulomatous Disease, Chronic, International Journal of Nanomedicine, NADPH oxidase complex, law, protein therapy, Drug Discovery, MESH: Phagocytosis, Candida, Original Research, Oxidase test, NADPH oxidase, [SDV.BBM.BS]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Structural Biology [q-bio.BM], biology, MESH: Reactive Oxygen Species, Cell Differentiation, Genetic Diseases, X-Linked, General Medicine, MESH: Protein Subunits, 3. Good health, medicine.anatomical_structure, Child, Preschool, Recombinant DNA, Nicotinamide adenine dinucleotide phosphate, MESH: Proteolipids, MESH: Cell Differentiation, induced pluripotent stem cells, Proteolipids, Biophysics, Bioengineering, MESH: Induced Pluripotent Stem Cells, MESH: NADPH Oxidases, Biomaterials, 03 medical and health sciences, Phagocytosis, proteoliposomes, MESH: Candida, MESH: Genetic Diseases, X-Linked, medicine, Humans, MESH: Humans, Macrophages, MESH: Child, Preschool, Organic Chemistry, Cell Membrane, MESH: Macrophages, NADPH Oxidases, medicine.disease, Molecular biology, Protein Subunits, 030104 developmental biology, chemistry, Membrane protein, Immunology, biology.protein, Reactive Oxygen Species, MESH: Cell Membrane
Abstract

Julie Brault,1,2 Guillaume Vaganay,3 Aline Le Roy,4–6 Jean-Luc Lenormand,1 Sandra Cortes,3 Marie José Stasia1,2 1UMR CNRS 5525, University of Grenoble Alpes, Grenoble, France; 2CGD Diagnosis and Research Centre, University Hospital Centre of Grenoble Alpes, Grenoble, France; 3Synthelis SAS, La Tronche, France; 4IBS, University of Grenoble Alpes, Grenoble, France; 5CNRS, IBS, University Grenoble Alpes, Grenoble, France; 6CEA, IBS, University of Grenoble Alpes, Grenoble, France Abstract: Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency due to dysfunction of the phagocytic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex leading to severe and recurrent infections in early childhood. The main genetic form is the X-linked CGD leading to the absence of cytochrome b558 composed of NOX2 and p22phox, the membrane partners of the NADPH oxidase complex. The first cause of death of CGD patients is pulmonary infections. Recombinant proteoliposome-based therapy is an emerging and innovative approach for membrane protein delivery, which could be an alternative local, targeted treatment to fight lung infections in CGD patients. We developed an enzyme therapy using recombinant NOX2/p22phox liposomes to supply the NADPH oxidase activity in X0-linked CGD (X0-CGD) macrophages. Using an optimized prokaryotic cell-free protein synthesis system, a recombinant cytochrome b558 containing functional hemes was produced and directly inserted into the lipid bilayer of specific liposomes. The size of the NOX2/p22phox liposomes was estimated to be around 700nm. These proteoliposomes were able to generate reactive oxygen species (ROS) in an activated reconstituted cell-free NADPH oxidase activation assay in the presence of recombinant p47phox, p67phox and Rac, the cytosolic components of the NADPH oxidase complex. Furthermore, using flow cytometry and fluorescence microscopy, we demonstrated that cytochrome b558 was successfully delivered to the plasma membrane of X0-CGD-induced pluripotent stem cell (iPSC)-derived macrophages. In addition, NADPH oxidase activity was restored in X0-CGD iPSC-derived macrophages treated with NOX2/p22phox liposomes for 8h without any toxicity. In conclusion, we confirmed that proteoliposomes provide a new promising technology for the delivery of functional proteins to the membrane of targeted cells. This efficient liposomal enzyme replacement therapy will be useful for future treatment of pulmonary infections in CGD patients refractory to conventional anti-infectious treatments. Keywords: protein therapy, proteoliposomes, chronic granulomatous disease, NADPH oxidase, induced pluripotent stem cells, macrophages&nbsp

Published
2017

13. ALS-causing mutations differentially affect PGC-1α expression and function in the brain vs. peripheral tissues

Author

Anke Witting, Eva Buck, Tanja Lucas, Jasmin Sprissler, Karin M Danzer, Karlheinz Holzmann, Kerstin Lang, Lara Barteczko, Tobias M. Boeckers, Patrick Weydt, Maria Demestre, Luc Dupuis, Hanna Bayer, Katrin S. Lindenberg, Noemi Pasquarelli, Julia Higelin, Albert C. Ludolph, University of Ulm (UUlm), Mécanismes Centraux et Périphériques de la Neurodégénérescence, Université de Strasbourg (UNISTRA)-Institut National de la Santé et de la Recherche Médicale (INSERM), University of Bonn, and Dieterle, Stéphane

Subjects
0301 basic medicine, [SDV]Life Sciences [q-bio], MESH: Neurons, PGC-1α, MESH: Protein Isoforms, [SCCO]Cognitive science, Superoxide Dismutase-1, 0302 clinical medicine, Adipose Tissue, Brown, Neurotrophic factors, Protein Isoforms, MESH: Animals, Amyotrophic lateral sclerosis, Induced pluripotent stem cell, MESH: Amyotrophic Lateral Sclerosis, MESH: Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha, MESH: Superoxide Dismutase-1, Neurons, MESH: Muscle, Skeletal, MESH: RNA-Binding Protein FUS, Neurodegeneration, Brain, SOD1, Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha, [SDV] Life Sciences [q-bio], Neurology, PPARGC1A, MESH: Mutation, MESH: Rats, MESH: Mice, Transgenic, Induced Pluripotent Stem Cells, Mice, Transgenic, MESH: Zebrafish Proteins, Biology, MESH: Induced Pluripotent Stem Cells, Neuroprotection, MESH: Adipose Tissue, Brown, Cell Line, lcsh:RC321-571, 03 medical and health sciences, MESH: Brain, Lou Gehrig disease, MESH: Mice, Inbred C57BL, Coactivator, medicine, Animals, Humans, RNA, Messenger, Muscle, Skeletal, MESH: Zebrafish, lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry, FUS, MESH: RNA, Messenger, MESH: Humans, [SCCO] Cognitive science, medicine.disease, Rats, MESH: Cell Line, Mice, Inbred C57BL, Disease Models, Animal, 030104 developmental biology, Mutation, Cancer research, RNA-Binding Protein FUS, Lactate, MESH: Disease Models, Animal, 030217 neurology & neurosurgery
Abstract

International audience; Background: Monogenetic forms of amyotrophic lateral sclerosis (ALS) offer an opportunity for unraveling the molecular mechanisms underlying this devastating neurodegenerative disorder. In order to identify a link between ALS-related metabolic changes and neurodegeneration, we investigated whether ALS-causing mutations interfere with the peripheral and brain-specific expression and signaling of the metabolic master regulator PGC (PPAR gamma coactivator)-1α (PGC-1α).Methods: We analyzed the expression of PGC-1α isoforms and target genes in two mouse models of familial ALS and validated the stimulated PGC-1α signaling in primary adipocytes and neurons of these animal models and in iPS derived motoneurons of two ALS patients harboring two different frame-shift FUS/TLS mutations.Results: Mutations in SOD1 and FUS/TLS decrease Ppargc1a levels in the CNS whereas in muscle and brown adipose tissue Ppargc1a mRNA levels were increased. Probing the underlying mechanism in neurons, we identified the monocarboxylate lactate as a previously unrecognized potent and selective inducer of the CNS-specific PGC-1α isoforms. Lactate also induced genes like brain-derived neurotrophic factor, transcription factor EB and superoxide dismutase 3 that are down-regulated in PGC-1α deficient neurons. The lactate-induced CNS-specific PGC-1α signaling system is completely silenced in motoneurons derived from induced pluripotent stem cells obtained from two ALS patients harboring two different frame-shift FUS/TLS mutations.Conclusion: ALS mutations increase the canonical PGC-1α system in the periphery while inhibiting the CNS-specific isoforms. We identify lactate as an inducer of the neuronal PGC-1α system directly linking brain metabolism and neuroprotection. Changes in the PGC-1α system might be involved in the ALS accompanied metabolic changes and in neurodegeneration.

Published
2017

14. Three-dimensional Quantification of Dendritic Spines from Pyramidal Neurons Derived from Human Induced Pluripotent Stem Cells

Author

Isabelle Cloëz-Tayarani, Thomas Bourgeron, Alexandra Benchoua, Laura Gouder, Hany Goubran-Botros, Jean-Yves Tinevez, Université Paris Diderot - Paris 7 (UPD7), Gènes, Synapses et Cognition (CNRS - UMR3571 ), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Génétique humaine et fonctions cognitives - Human Genetics and Cognitive Functions (GHFC (UMR_3571 / U-Pasteur_1)), Institut Pasteur [Paris] (IP)-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Imagerie Dynamique (Plate-Forme) (PFID), Institut Pasteur [Paris] (IP), Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, This work was funded by the Institut Pasteur, the Bettencourt-Schueller foundation, Centre National de la Recherche Scientifique, University Paris Diderot, Agence Nationale de la Recherche (ANR-13-SAMA-0006, SynDivAutism), the Conny-Maeva Charitable Foundation, the Cognacq Jay Foundation, the Orange Foundation, and the Fondamental Foundation. L.G. is supported by an undergraduate fellowship from the Health Ministry., We acknowledge the help of BitPlane in particular Georgia Golfis, in the early stage of this work., ANR-13-SAMA-0006,SynDivAutism,Diversité Synaptique dans l'autisme(2013), Gènes, Synapses et Cognition, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris]-Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris], and Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)

Subjects
Pluripotent Stem Cells, MESH: Neural Stem Cells, Dendritic spine, General Chemical Engineering, Dendritic Spines, [SDV]Life Sciences [q-bio], Green Fluorescent Proteins, Induced Pluripotent Stem Cells, Glutamic Acid, Biology, MESH: Induced Pluripotent Stem Cells, MESH: Dendrites, Receptors, N-Methyl-D-Aspartate, General Biochemistry, Genetics and Molecular Biology, MESH: Synapses, MESH: Dendritic Spines, Glutamatergic, [SCCO]Cognitive science, MESH: Green Fluorescent Proteins, Neural Stem Cells, Postsynaptic potential, medicine, Premovement neuronal activity, Humans, MESH: Microscopy, Confocal, Induced pluripotent stem cell, MESH: Receptors, N-Methyl-D-Aspartate, [SDV.GEN]Life Sciences [q-bio]/Genetics, Microscopy, Confocal, MESH: Humans, General Immunology and Microbiology, General Neuroscience, Pyramidal Cells, Dendrites, MESH: Glutamic Acid, MESH: Pyramidal Cells, Neural stem cell, medicine.anatomical_structure, [SDV.MHEP.PSM]Life Sciences [q-bio]/Human health and pathology/Psychiatrics and mental health, Synapses, MESH: Pluripotent Stem Cells, Neuron, Stem cell, Neuroscience, Developmental Biology
Abstract

International audience; Dendritic spines are small protrusions that correspond to the post-synaptic compartments of excitatory synapses in the central nervous system. They are distributed along the dendrites. Their morphology is largely dependent on neuronal activity, and they are dynamic. Dendritic spines express glutamatergic receptors (AMPA and NMDA receptors) on their surface and at the levels of postsynaptic densities. Each spine allows the neuron to control its state and local activity independently. Spine morphologies have been extensively studied in glutamatergic pyramidal cells of the brain cortex, using both in vivo approaches and neuronal cultures obtained from rodent tissues. Neuropathological conditions can be associated to altered spine induction and maturation, as shown in rodent cultured neurons and one-dimensional quantitative analysis (1). The present study describes a protocol for the 3D quantitative analysis of spine morphologies using human cortical neurons derived from neural stem cells (late cortical progenitors). These cells were initially obtained from induced pluripotent stem cells. This protocol allows the analysis of spine morphologies at different culture periods, and with possible comparison between induced pluripotent stem cells obtained from control individuals with those obtained from patients with psychiatric diseases.

Published
2015

15. Human induced pluripotent stem cells in hepatology: beyond the proof of concept

Author

Gerbal-Chaloin, Sabine, Funakoshi, Natalie, Caillaud, Amandine, Gondeau, Claire, Champon, Benoite, Si-Tayeb, Karim, Cellules souches normales et cancéreuses, Université Montpellier 1 (UM1)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Montpellier (UM), Service d'Hépato-gastro-entérologie B, Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-CHU Saint-Eloi, unité de recherche de l'institut du thorax UMR1087 UMR6291 (ITX), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), Université de Nantes (UN)-Université de Nantes (UN), Equipe 5 : Investigation moléculaires dans les dyslipidémies, Université de Nantes (UN)-Université de Nantes (UN)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université de Nantes - UFR de Médecine et des Techniques Médicales (UFR MEDECINE), European Project: 277188,EC:FP7:PEOPLE,FP7-PEOPLE-2010-RG,IPSMILD(2011), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier)-Hôpital Saint Eloi (CHRU Montpellier), Centre Hospitalier Régional Universitaire [Montpellier] (CHRU Montpellier), Unité de recherche de l'institut du thorax (ITX-lab), Si-Tayeb, Karim, and HUMAN INDUCED PLURIPOTENT STEM CELLS AS A MODEL TO STUDY METABOLIC INHERITED LIVER DISEASES - IPSMILD - - EC:FP7:PEOPLE2011-12-01 - 2014-11-30 - 277188 - VALID

Subjects
MESH: Cell Differentiation, MESH: Nuclear Reprogramming, MESH: Humans, Induced Pluripotent Stem Cells, Gastroenterology, Cell Differentiation, [SDV.MHEP.HEG]Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, MESH: Gastroenterology, MESH: Induced Pluripotent Stem Cells, Cellular Reprogramming, [SDV.MHEP.HEG] Life Sciences [q-bio]/Human health and pathology/Hépatology and Gastroenterology, MESH: Hepatocytes, Hepatocytes, Animals, Humans, MESH: Animals, MESH: Stem Cell Transplantation, Stem Cell Transplantation
Abstract

International audience; The discovery of the wide plasticity of most cell types means that it is now possible to produce virtually any cell type in vitro. This concept, developed because of the possibility of reprogramming somatic cells toward induced pluripotent stem cells, provides the opportunity to produce specialized cells that harbor multiple phenotypical traits, thus integrating genetic interindividual variability. The field of hepatology has exploited this concept, and hepatocyte-like cells can now be differentiated from induced pluripotent stem cells. This review discusses the choice of somatic cells to be reprogrammed by emergent new and nonintegrative strategies, as well as the application of differentiated human induced pluripotent stem cells in hepatology, including liver development, disease modeling, host-pathogen interactions, and drug metabolism and toxicity. The actual consensus is that hepatocyte-like cells generated in vitro present an immature phenotype. Currently, developed strategies used to resolve this problem, such as overexpression of transcription factors, mimicking liver neonatal and postnatal modifications, and re-creating the three-dimensional hepatocyte environment in vitro and in vivo, are also discussed.

Published
2014

16. Modeling neuronal defects associated with a lysosomal disorder using patient-derived induced pluripotent stem cells

Author

Diana Toli, Elise Roy, Thomas Lemonnier, Isabelle Rouvet, Delphine Bohl, Stéphane Blanchard, Stéphanie Bigou, Jean Michel Heard, Sandrine Vitry, Roseline Froissart, Rétrovirus et Transfert Génétique, Institut Pasteur [Paris]-Institut National de la Santé et de la Recherche Médicale (INSERM), Centre de Biologie et Pathologie Est (CBPE), Hospices Civils de Lyon (HCL)-Centre National de Référence des Légionelles, Laboratoire des Maladies Héréditaires du Métabolisme, Hospices Civils de Lyon (HCL)-CHU de Lyon-GH Est-Centre de Biologie et de Pathologie Est - CBPE, Centre de Biotechnologie cellulaire, Hospices Civils de Lyon (HCL), Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Institut Pasteur [Paris] (IP)-Institut National de la Santé et de la Recherche Médicale (INSERM), Groupement Hospitalier Lyon-Est (GHE), Hospices Civils de Lyon (HCL)-Hospices Civils de Lyon (HCL)-Centre de Biologie et de Pathologie Est - CBPE, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Male, MESH: Acetylglucosaminidase, [SDV]Life Sciences [q-bio], MESH: Neurons, Fibroblast growth factor, Mucopolysaccharidosis III, 0302 clinical medicine, MESH: Child, Lysosomal storage disease, Induced pluripotent stem cell, Child, MESH: Mucopolysaccharidosis III, Genetics (clinical), Cells, Cultured, Neurons, 0303 health sciences, Cell Differentiation, General Medicine, Neural stem cell, Cell biology, medicine.anatomical_structure, Child, Preschool, symbols, Female, [SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC], Stem cell, MESH: Cells, Cultured, MESH: Cell Differentiation, MESH: Mutation, Induced Pluripotent Stem Cells, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, MESH: Induced Pluripotent Stem Cells, Models, Biological, 03 medical and health sciences, symbols.namesake, Lysosome, Neurosphere, MESH: Cell Proliferation, Acetylglucosaminidase, Genetics, medicine, Humans, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Molecular Biology, 030304 developmental biology, Cell Proliferation, MESH: Humans, MESH: Child, Preschool, MESH: Models, Biological, Golgi apparatus, Fibroblasts, medicine.disease, MESH: Male, MESH: Fibroblasts, Mutation, MESH: Heparan Sulfate Proteoglycans, Lysosomes, MESH: Female, 030217 neurology & neurosurgery, Heparan Sulfate Proteoglycans, MESH: Lysosomes
Abstract

International audience; By providing access to affected neurons, human induced pluripotent stem cells (iPSc) offer a unique opportunity to model human neurodegenerative diseases. We generated human iPSc from the skin fibroblasts of children with mucopolysaccharidosis type IIIB. In this fatal lysosomal storage disease, defective α-N-acetylglucosaminidase interrupts the degradation of heparan sulfate (HS) proteoglycans and induces cell disorders predominating in the central nervous system, causing relentless progression toward severe mental retardation. Partially digested proteoglycans, which affect fibroblast growth factor signaling, accumulated in patient cells. They impaired isolation of emerging iPSc unless exogenous supply of the missing enzyme cleared storage and restored cell proliferation. After several passages, patient iPSc starved of an exogenous enzyme continued to proliferate in the presence of fibroblast growth factor despite HS accumulation. Survival and neural differentiation of patient iPSc were comparable with unaffected controls. Whereas cell pathology was modest in floating neurosphere cultures, undifferentiated patient iPSc and their neuronal progeny expressed cell disorders consisting of storage vesicles and severe disorganization of Golgi ribbons associated with modified expression of the Golgi matrix protein GM130. Gene expression profiling in neural stem cells pointed to alterations of extracellular matrix constituents and cell-matrix interactions, whereas genes associated with lysosome or Golgi apparatus functions were downregulated. Taken together, these results suggest defective responses of patient undifferentiated stem cells and neurons to environmental cues, which possibly affect Golgi organization, cell migration and neuritogenesis. This could have potential consequences on post-natal neurological development, once HS proteoglycan accumulation becomes prominent in the affected child brain.

Published
2011

17. A purified population of multipotent cardiovascular progenitors derived from primate pluripotent stem cells engrafts in postmyocardial infarcted nonhuman primates

Author

Blin , Guillaume, Nury , David, Stefanovic , Sonia, Neri , Tui, Guillevic , Orianne, Brinon , Benjamin, Bellamy , Valérie, Rücker-Martin , Catherine, Barbry , Pascal, Bel , Alain, Bonnevie , Lionel, Bruneval , Patrick, Cowan , Chad, Pouly , Julia, Mitalipov , Shoukhrat, Gouadon , Elodie, Binder , Patrice, Hagège , Albert, Desnos , Michel, Menasché , Philippe, Renaud , Jean-François, Puceat , Michel, Institut des cellules souches pour le traitement et l'étude des maladies monogéniques ( I-STEM ), Université d'Évry-Val-d'Essonne ( UEVE ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ), Therapie Cellulaire en Pathologie Cardio-Vasculaire ( UMR_S 633 ), Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Université Paris Descartes - Paris 5 ( UPD5 ), Remodelage tissulaire et fonctionnel : signalisation et physio-pathologie ( RTFSPP ), Université Paris-Sud - Paris 11 ( UP11 ) -Centre chirurgical Marie Lannelongue-Centre National de la Recherche Scientifique ( CNRS ), Institut de pharmacologie moléculaire et cellulaire ( IPMC ), Université Nice Sophia Antipolis ( UNS ), Université Côte d'Azur ( UCA ) -Université Côte d'Azur ( UCA ) -Centre National de la Recherche Scientifique ( CNRS ), Service de chirurgie cardiaque et vasculaire [CHU HEGP], Assistance publique - Hôpitaux de Paris (AP-HP)-Hôpital Européen Georges Pompidou [APHP] ( HEGP ), Service de cardiologie, Hôpital d'Instruction des Armées Bégin, Service d'anatomo-pathologie [CHU HEGP], Stowers Medical Institute, Cardiovascular Research Center, Division of Reproductive Sciences, Oregon National Primate Research Center, Institut de Médecine Aérospatiale du Service de Santé des Armées ( IMASSA ), Service de Santé des Armées, Service de cardiologie [CHU HEGP], Equipe avenir HUMAN EMBRYONIC STEM CELLS AS A MODEL TO DELINEATE THE EARLY CARDIAC TRANSCRIPTIONAL NETWORKS: TOWARD A BETTER UNDERSTANDING OF CARDIAC CONGENITAL AND GENETIC DISEASES., Institut National de la Santé et de la Recherche Médicale ( INSERM ), Guillaume Blin is a fellow from the Ministère de la Recherche et de la Technologie, David Nury and Sonia Stefanovic were funded by the Grants « programme Blanc stem cell signature » and 'Specistem' of the National Research Agency (to MP). This study was funded by the National Research Agency (grants Programmes Blanc stem cell signature and Maladies rares to MP ), the Genopole Evry, the French Association against Myopathies (AFM grant n°13968 to CR), the LeDucq Foundation (CAPTAA) (Paris to PM and MP), the Foundation Coeur et Artères and the Fonds d'Amorçage des Biothérapies (AP-HP, to PM). Tui Neri is a fellow from the LeDucq Foundation (MITRAL to MP)., ANR-07-BLAN-0058,BLANC,Determinants of hematopoietic stem cell / niche interactions in lineage commitment ( 2007 ), Institut des cellules souches pour le traitement et l'étude des maladies monogéniques (I-STEM), Université d'Évry-Val-d'Essonne (UEVE)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Généthon, Therapie Cellulaire en Pathologie Cardio-Vasculaire (UMR_S 633), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM), Remodelage tissulaire et fonctionnel : signalisation et physio-pathologie (RTFSPP), Université Paris-Sud - Paris 11 (UP11)-Centre Chirurgical Marie Lannelongue (CCML)-Centre National de la Recherche Scientifique (CNRS), Institut de pharmacologie moléculaire et cellulaire (IPMC), Université Nice Sophia Antipolis (1965 - 2019) (UNS), COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-COMUE Université Côte d'Azur (2015-2019) (COMUE UCA)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Européen Georges Pompidou [APHP] (HEGP), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO)-Hôpitaux Universitaires Paris Ouest - Hôpitaux Universitaires Île de France Ouest (HUPO), Oregon National Primate Research Center (ONPRC), Oregon Health and Science University [Portland] (OHSU)-Oregon Health and Science University [Portland] (OHSU), Institut de Médecine Aérospatiale du Service de Santé des Armées (IMASSA), Institut National de la Santé et de la Recherche Médicale (INSERM), ANR-07-BLAN-0058,HSC-FATE,Determinants of hematopoietic stem cell / niche interactions in lineage commitment(2007), Centre National de la Recherche Scientifique (CNRS)-Centre chirurgical Marie Lannelongue-Université Paris-Sud - Paris 11 (UP11), Université Nice Sophia Antipolis (... - 2019) (UNS), Puceat, Michel, and Blanc - Determinants of hematopoietic stem cell / niche interactions in lineage commitment - - HSC-FATE2007 - ANR-07-BLAN-0058 - BLANC - VALID

Subjects
Pathology, MESH: Myocytes, Cardiac, MESH: Bone Morphogenetic Protein 2, 030204 cardiovascular system & hematology, 0302 clinical medicine, MESH: Animals, Induced pluripotent stem cell, MESH: Embryonic Stem Cells, Stem cell transplantation for articular cartilage repair, MESH: Octamer Transcription Factor-3, 0303 health sciences, Induced stem cells, MESH: Antigens, CD15, General Medicine, [ SDV.MHEP.CSC ] Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, [SDV.MHEP.CSC] Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, 3. Good health, Cell biology, MESH: Myocardial Infarction, MESH : Antigens, CD31, embryonic structures, Stem cell, MESH: Stem Cell Transplantation, MESH : Macaca mulatta, MESH : Cell Differentiation, Adult stem cell, MESH: Cells, Cultured, MESH : Induced Pluripotent Stem Cells, MESH: Cell Differentiation, medicine.medical_specialty, MESH : Multipotent Stem Cells, MESH : Myocytes, Cardiac, Clinical uses of mesenchymal stem cells, Biology, MESH: Induced Pluripotent Stem Cells, MESH : Stem Cell Transplantation, MESH: Macaca mulatta, 03 medical and health sciences, [SDV.MHEP.CSC]Life Sciences [q-bio]/Human health and pathology/Cardiology and cardiovascular system, MESH : Cells, Cultured, medicine, MESH : Embryonic Stem Cells, Progenitor cell, 030304 developmental biology, MESH: Humans, MESH : Octamer Transcription Factor-3, MESH : Humans, MESH: Antigens, CD31, Embryonic stem cell, MESH : MicroRNAs, MESH : Antigens, CD15, MESH : Animals, MESH: Multipotent Stem Cells, MESH : Myocardial Infarction, MESH : Bone Morphogenetic Protein 2, MESH: MicroRNAs
Abstract

International audience; Cell therapy holds promise for tissue regeneration, including in individuals with advanced heart failure. However, treatment of heart disease with bone marrow cells and skeletal muscle progenitors has had only marginal positive benefits in clinical trials, perhaps because adult stem cells have limited plasticity. The identification, among human pluripotent stem cells, of early cardiovascular cell progenitors required for the development of the first cardiac lineage would shed light on human cardiogenesis and might pave the way for cell therapy for cardiac degenerative diseases. Here, we report the isolation of an early population of cardiovascular progenitors, characterized by expression of OCT4, stage-specific embryonic antigen 1 (SSEA-1), and mesoderm posterior 1 (MESP1), derived from human pluripotent stem cells treated with the cardiogenic morphogen BMP2. This progenitor population was multipotential and able to generate cardiomyocytes as well as smooth muscle and endothelial cells. When transplanted into the infarcted myocardium of immunosuppressed nonhuman primates, an SSEA-1+ progenitor population derived from Rhesus embryonic stem cells differentiated into ventricular myocytes and reconstituted 20% of the scar tissue. Notably, primates transplanted with an unpurified population of cardiac-committed cells, which included SSEA-1- cells, developed teratomas in the scar tissue, whereas those transplanted with purified SSEA-1+ cells did not. We therefore believe that the SSEA-1+ progenitors that we have described here have the potential to be used in cardiac regenerative medicine.

Published
2010

18. Creating a blood line from human skin

Author

Michael H. Sieweke, Centre d'Immunologie de Marseille - Luminy (CIML), Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Aix Marseille Université (AMU), and Aix Marseille Université (AMU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)

Subjects
MESH: Cell Differentiation, Lineage (genetic), Cellular differentiation, Induced Pluripotent Stem Cells, Cell Culture Techniques, Human skin, Biology, MESH: Induced Pluripotent Stem Cells, Cell Line, Blood cell, 03 medical and health sciences, MESH: Skin, medicine, Humans, Cell Lineage, Progenitor cell, Induced pluripotent stem cell, Skin, 030304 developmental biology, 0303 health sciences, MESH: Cell Culture Techniques, Blood Cells, MESH: Humans, MESH: Blood Cells, 030302 biochemistry & molecular biology, Cell Differentiation, Fibroblasts, MESH: Cell Lineage, Research Highlight, Cell biology, MESH: Cell Line, medicine.anatomical_structure, Cell culture, MESH: Fibroblasts, [SDV.IMM]Life Sciences [q-bio]/Immunology, Reprogramming
Abstract

International audience; ABSTRACT : A recent study has generated blood cell progenitors with therapeutic potential by direct lineage conversion of human fibroblasts, thus circumventing reprogramming to pluripotent stem cells.

Published
2010

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