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1.
Mol Cell ; 58(6): 1001-14, 2015 Jun 18.
Article in English | MEDLINE | ID: mdl-26004228

ABSTRACT

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein that, beyond its apoptotic function, is required for the normal expression of major respiratory chain complexes. Here we identified an AIF-interacting protein, CHCHD4, which is the central component of a redox-sensitive mitochondrial intermembrane space import machinery. Depletion or hypomorphic mutation of AIF caused a downregulation of CHCHD4 protein by diminishing its mitochondrial import. CHCHD4 depletion sufficed to induce a respiratory defect that mimicked that observed in AIF-deficient cells. CHCHD4 levels could be restored in AIF-deficient cells by enforcing its AIF-independent mitochondrial localization. This modified CHCHD4 protein reestablished respiratory function in AIF-deficient cells and enabled AIF-deficient embryoid bodies to undergo cavitation, a process of programmed cell death required for embryonic morphogenesis. These findings explain how AIF contributes to the biogenesis of respiratory chain complexes, and they establish an unexpected link between the vital function of AIF and the propensity of cells to undergo apoptosis.


Subject(s)
Apoptosis Inducing Factor/metabolism , Electron Transport Chain Complex Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/metabolism , Amino Acid Sequence , Animals , Apoptosis Inducing Factor/genetics , Cell Line, Tumor , Electron Transport/genetics , Electron Transport Chain Complex Proteins/genetics , Embryo, Mammalian/embryology , Embryo, Mammalian/metabolism , Embryonic Development/genetics , Humans , Immunoblotting , Mice, Knockout , Mitochondria/genetics , Mitochondrial Membrane Transport Proteins/genetics , Mitochondrial Precursor Protein Import Complex Proteins , Molecular Sequence Data , Protein Binding , Protein Transport/genetics , RNA Interference , Time Factors
2.
Dev Biol ; 461(1): 86-95, 2020 05 01.
Article in English | MEDLINE | ID: mdl-31982375

ABSTRACT

One of the main obstacles for studying the molecular and cellular mechanisms underlying human neurodevelopment in vivo is the scarcity of experimental models. The discovery that neurons can be generated from human induced pluripotent stem cells (hiPSCs) paves the way for novel approaches that are stem cell-based. Here, we developed a technique to follow the development of transplanted hiPSC-derived neuronal precursors in the cortex of mice over time. Using post-mortem immunohistochemistry we quantified the differentiation and maturation of dendritic patterns of the human neurons over a total of six months. In addition, entirely hiPSC-derived neuronal parenchyma was followed over eight months using two-photon in vivo imaging through a cranial window. We found that transplanted hiPSC-derived neuronal precursors exhibit a "protracted" human developmental programme in different cortical areas. This offers novel possibilities for the sequential in vivo study of human cortical development and its alteration, followed in "real time".


Subject(s)
Induced Pluripotent Stem Cells/transplantation , Motor Cortex/embryology , Neurogenesis/physiology , Pyramidal Cells/transplantation , Animals , Brain/embryology , Cells, Cultured , Humans , Induced Pluripotent Stem Cells/cytology , Mice , Mice, Inbred NOD , Mice, SCID , Motor Cortex/cytology , Pyramidal Cells/cytology , Transplantation, Heterologous
3.
Int J Mol Sci ; 20(7)2019 Apr 08.
Article in English | MEDLINE | ID: mdl-30965622

ABSTRACT

Although human pluripotent stem cells (hPSCs) can theoretically differentiate into any cell type, their ability to produce hematopoietic cells is highly variable from one cell line to another. The underlying mechanisms of this heterogeneity are not clearly understood. Here, using a whole miRNome analysis approach in hPSCs, we discovered that their hematopoietic competency was associated with the expression of several miRNAs and conversely correlated to that of miR-206 specifically. Lentiviral-based miR-206 ectopic expression in H1 hematopoietic competent embryonic stem (ES) cells markedly impaired their differentiation toward the blood lineage. Integrative bioinformatics identified a potential miR-206 target gene network which included hematopoietic master regulators RUNX1 and TAL1. This work sheds light on the critical role of miR-206 in the generation of blood cells off hPSCs. Our results pave the way for future genetic manipulation of hPSCs aimed at increasing their blood regenerative potential and designing better protocols for the generation of bona fide hPSC-derived hematopoietic stem cells.


Subject(s)
MicroRNAs/metabolism , Pluripotent Stem Cells/cytology , Cell Differentiation/physiology , Cell Line , Cell Lineage , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/metabolism , Humans , Pluripotent Stem Cells/metabolism
4.
Int J Mol Sci ; 20(19)2019 Sep 30.
Article in English | MEDLINE | ID: mdl-31575031

ABSTRACT

Hereditary cancers with cancer-predisposing mutations represent unique models of human oncogenesis, as a driving oncogenic event is present in germline. Currently, there are no satisfactory models to study these malignancies. We report the generation of IPSC from the somatic cells of a patient with hereditary c-met-mutated papillary renal cell carcinoma (PRCC). From these cells we have generated spontaneous aggregates organizing in structures which expressed kidney markers such as PODXL and Six2. These structures expressed PRCC markers both in vitro and in vivo in NSG mice. Gene-expression profiling showed striking molecular similarities with signatures found in a large cohort of PRCC tumor samples. This analysis, applied to primary cancers with and without c-met mutation, showed overexpression of the BHLHE40 and KDM4C only in the c-met-mutated PRCC tumors, as predicted by c-met-mutated embryoid bodies transcriptome. These data therefore represent the first proof of concept of "hereditary renal cancer in a dish" model using c-met-mutated iPSC-derived embryoid bodies, opening new perspectives for discovery of novel predictive progression markers and for drug-screening for future precision-medicine strategies.


Subject(s)
Carcinoma, Papillary/etiology , Carcinoma, Renal Cell/etiology , Embryoid Bodies/cytology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Mutation , Proto-Oncogene Proteins c-met/genetics , Alleles , Carcinoma, Papillary/diagnosis , Carcinoma, Renal Cell/diagnosis , Embryoid Bodies/metabolism , Embryoid Bodies/ultrastructure , Fluorescent Antibody Technique , Gene Expression , Genotype , Humans , Immunohistochemistry , Magnetic Resonance Imaging/methods , Reproducibility of Results
5.
Biochem Biophys Res Commun ; 503(3): 1861-1867, 2018 09 10.
Article in English | MEDLINE | ID: mdl-30057314

ABSTRACT

Despite the major success obtained by the use of tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML), resistances to therapies occur due to mutations in the ABL-kinase domain of the BCR-ABL oncogene. Amongst these mutations, the "gatekeeper" T315I is a major concern as it renders leukemic cells resistant to all licenced TKI except Ponatinib. We report here that Fourier transform infrared (FTIR) microspectroscopy is a powerful methodology allowing rapid and direct identification of a spectral signature in single cells expressing T315I-mutated BCR-ABL. The specificity of this spectral signature is confirmed using a Dox-inducible T315I-mutated BCR-ABL-expressing human UT-7 cells as well as in murine embryonic stem cells. Transcriptome analysis of UT-7 cells expressing BCR-ABL as compared to BCR-ABL T315I clearly identified a molecular signature which could be at the origin of the generation of metabolic changes giving rise to the spectral signature. Thus, these results suggest that this new methodology can be applied to the identification of leukemic cells harbouring the T315I mutation at the single cell level and could represent a novel early detection tool of mutant clones. It could also be applied to drug screening strategies to target T315I-mutated leukemic cells.


Subject(s)
Fusion Proteins, bcr-abl/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Spectroscopy, Fourier Transform Infrared , Animals , Cell Line , Fusion Proteins, bcr-abl/genetics , Humans , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/pathology , Mice , Mutation
6.
Am J Pathol ; 180(5): 2084-96, 2012 May.
Article in English | MEDLINE | ID: mdl-22425713

ABSTRACT

Because many of the genes used to produce induced pluripotent stem cells (iPSCs) from somatic cells are either outright established oncogenes, such as c-myc and Klf4, or potentially related to tumorigenesis in various cancers, both the safety and the risks of tumorigenesis linked to iPSC generation require evaluation. In this work, we generated, by lentivirus-mediated gene transfer of Oct4, Sox2, Nanog, and Lin28, two types of iPSCs from human mesenchymal stem cells and human amniotic fluid-derived cells: fully reprogrammed iPSCs with silencing of the four transgenes and partially reprogrammed iPSCs that still express one or several transgenes. We assessed the behavior of these cells during both their differentiation and proliferation using in vivo teratoma assays in nonobese diabetic mice with severe combined immunodeficiency. In contrast to fully reprogrammed iPSCs, 43% of partially reprogrammed iPSC cases (6 of 14 teratomas) generated major dysplasia and malignant tumors, with yolk sac tumors and embryonal carcinomas positive for α-fetoprotein, cytokeratin AE1/AE3, and CD30. This correlated with the expression of one or several transgenes used for the reprogramming, down-regulation of CDK 1A mRNA (p21/CDKN1A), and up-regulation of antiapoptotic Bcl-2 mRNA. Therefore, the oncogenicity of therapeutically valuable patient-specific iPSC-derived cells should be scrupulously evaluated before they are used for any clinical applications.


Subject(s)
Cell Transformation, Neoplastic/pathology , Induced Pluripotent Stem Cells/pathology , Ki-1 Antigen/metabolism , Neoplasms, Germ Cell and Embryonal/pathology , Animals , Cell Differentiation/physiology , Cell Proliferation , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Cellular Reprogramming/physiology , Embryonic Stem Cells/cytology , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , Karyotype , Kruppel-Like Factor 4 , Mice , Mice, Inbred NOD , Mice, SCID , Neoplasms, Germ Cell and Embryonal/genetics , Neoplasms, Germ Cell and Embryonal/metabolism , Teratoma/metabolism , Teratoma/pathology , Transgenes/genetics
7.
Blood ; 114(8): 1506-17, 2009 Aug 20.
Article in English | MEDLINE | ID: mdl-19478046

ABSTRACT

The megakaryocytic (MK) and erythroid lineages are tightly associated during differentiation and are generated from a bipotent megakaryocyte-erythroid progenitor (MEP). In the mouse, a primitive MEP has been demonstrated in the yolk sac. In human, it is not known whether the primitive MK and erythroid lineages are generated from a common progenitor or independently. Using hematopoietic differentiation of human embryonic stem cells on the OP9 cell line, we identified a primitive MEP in a subset of cells coexpressing glycophorin A (GPA) and CD41 from day 9 to day 12 of coculturing. This MEP differentiates into primitive erythroid (GPA(+)CD41(-)) and MK (GPA(-)CD41(+)) lineages. In contrast to erythropoietin (EPO)-dependent definitive hematopoiesis, KIT was not detected during erythroid differentiation. A molecular signature for the commitment and differentiation toward both the erythroid and MK lineages was detected by assessing expression of transcription factors, thrombopoietin receptor (MPL) and erythropoietin receptor (EPOR). We showed an inverse correlation between FLI1 and both KLF1 and EPOR during primitive erythroid and MK differentiation, similar to definitive hematopoiesis. This novel MEP differentiation system may allow an in-depth exploration of the molecular bases of erythroid and MK commitment and differentiation.


Subject(s)
Embryonic Stem Cells/physiology , Erythroid Cells , Hematopoiesis/physiology , Megakaryocyte-Erythroid Progenitor Cells/physiology , Megakaryocytes/physiology , Animals , Antigens, CD34/metabolism , Cell Differentiation/physiology , Cell Lineage/physiology , Cells, Cultured , Coculture Techniques , Embryonic Stem Cells/metabolism , Erythroid Cells/cytology , Erythroid Cells/metabolism , Glycophorins/metabolism , Humans , Leukosialin/metabolism , Megakaryocyte-Erythroid Progenitor Cells/metabolism , Megakaryocytes/metabolism , Mice , Platelet Glycoprotein GPIb-IX Complex/metabolism , Platelet Membrane Glycoprotein IIb/metabolism
8.
Apoptosis ; 15(12): 1529-39, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20640889

ABSTRACT

The protein Puma (p53-upregulated modulator of apoptosis) belongs to the BH3-only group of the Bcl-2 family and is a major regulator of apoptosis. Although the transcriptional regulation of Puma is clearly established, little is known about the regulation of its expression at the protein levels. We show here that various signals--including the cytokine TGFß, the death effector TRAIL or chemical drugs such as anisomycin--downregulate Puma protein levels via a novel pathway based on the sequential activation of caspase-3 and a protease inhibited by the serpase inhibitor N-tosyl-L-phenylalanine chloromethyl ketone. This pathway is specific for Puma because (1) the levels of other BH3-only proteins, such as Bim and Noxa were not modified by these stimuli and (2) this caspase-mediated degradation was dependent on both the BH3 and C-terminal domains of Puma. Our data also show that Puma is regulated during the caspase-3-dependent differentiation of murine embryonic stem cells and suggest that this pathway may be relevant and important during caspase-mediated cell differentiation not associated with apoptosis.


Subject(s)
Apoptosis Regulatory Proteins , Caspase 3 , Cell Differentiation/drug effects , Cell Differentiation/physiology , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Peptide Fragments/physiology , Proto-Oncogene Proteins/physiology , Serine Proteases , Signal Transduction/drug effects , Signal Transduction/physiology , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Tosylphenylalanyl Chloromethyl Ketone , Transforming Growth Factor beta/pharmacology , Animals , Anisomycin/pharmacology , Apoptosis Regulatory Proteins/genetics , Apoptosis Regulatory Proteins/metabolism , Caspase 3/metabolism , Cell Line, Tumor , Enzyme Inhibitors/pharmacology , Gene Silencing/physiology , Humans , Mice , Protein Structure, Tertiary , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Serine Proteases/metabolism , TNF-Related Apoptosis-Inducing Ligand/genetics , Tosylphenylalanyl Chloromethyl Ketone/pharmacology , Transforming Growth Factor beta/genetics
9.
Stem Cells ; 27(8): 1750-9, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19544443

ABSTRACT

Embryoid bodies (EBs) generated during differentiation of human embryonic stem cells (hESCs) contain vascular-like structures, suggesting that commitment of mesoderm progenitors into endothelial cells occurs spontaneously. We showed that bone morphogenetic protein 4 (BMP4), an inducer of mesoderm, accelerates the peak expression of CD133/kinase insert domain-containing receptor (KDR) and CD144/KDR. Because the CD133(+)KDR(+) population could represent endothelial progenitors, we sorted them at day 7 and cultured them in endothelial medium. These cells were, however, unable to differentiate into endothelial cells. Under standard conditions, the CD144(+)KDR(+) population represents up to 10% of the total cells at day 12. In culture, these cells, if sorted, give rise to a homogeneous population with a morphology typical of endothelial cells and express endothelial markers. These endothelial cells derived from the day 12 sorted population were functional, as assessed by different in vitro assays. When EBs were stimulated by BMP4, the CD144(+)KDR(+) peak was shifted to day 7. Most of these cells, however, were CD31(-), becoming CD31(+) in culture. They then expressed von Willebrand factor and were functional. This suggests that, initially, the BMP4-boosted day 7, CD144(+)KDR(+)CD31(-) population represents immature endothelial cells that differentiate into mature endothelial cells in culture. The expression of OCT3/4, a marker of immaturity for hESCs decreases during EB differentiation, decreasing faster following BMP4 induction. We also show that BMP4 inhibits the global expression of GATA2 and RUNX1, two transcription factors involved in hemangioblast formation, at day 7 and day 12.


Subject(s)
Bone Morphogenetic Protein 4/pharmacology , Embryonic Stem Cells/drug effects , Endothelial Cells/drug effects , Antigens, CD/biosynthesis , Cadherins/biosynthesis , Carrier Proteins/pharmacology , Cell Differentiation/drug effects , Cell Growth Processes/drug effects , Cell Growth Processes/physiology , Cells, Cultured , Cytokines/pharmacology , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Endothelial Cells/cytology , Endothelial Cells/metabolism , Flow Cytometry , Humans , Kinetics , Transcription Factors/biosynthesis
10.
Oncotarget ; 10(28): 2693-2708, 2019 Apr 12.
Article in English | MEDLINE | ID: mdl-31105870

ABSTRACT

Recent development of cell reprogramming technologies brought a major hope for future cell therapy applications by the use of these cells or their derivatives. For this purpose, one of the major requirements is the absence of genomic alterations generating a risk of cell transformation. Here we analyzed by microarray-based comparative genomic hybridization human iPSC generated by two non-integrative and one integrative method at pluripotent stage as well as in corresponding teratomas. We show that all iPSC lines exhibit copy number variations (CNV) of several genes deregulated in oncogenesis. These cancer-associated genomic alterations were more pronounced in virally programmed hiPSCs and their derivative teratoma as compared to those found in iPSC generated by mRNA-mediated reprogramming. Bioinformatics analysis showed the involvement of these genes in human leukemia and carcinoma. We conclude that genetic screening should become a standard procedure to ensure that hiPSCs are free from cancer-associated genomic alterations before clinical use.

11.
J Clin Invest ; 129(5): 2145-2162, 2019 04 15.
Article in English | MEDLINE | ID: mdl-30985297

ABSTRACT

Vacuolar H+-ATPase-dependent (V-ATPase-dependent) functions are critical for neural proteostasis and are involved in neurodegeneration and brain tumorigenesis. We identified a patient with fulminant neurodegeneration of the developing brain carrying a de novo splice site variant in ATP6AP2 encoding an accessory protein of the V-ATPase. Functional studies of induced pluripotent stem cell-derived (iPSC-derived) neurons from this patient revealed reduced spontaneous activity and severe deficiency in lysosomal acidification and protein degradation leading to neuronal cell death. These deficiencies could be rescued by expression of full-length ATP6AP2. Conditional deletion of Atp6ap2 in developing mouse brain impaired V-ATPase-dependent functions, causing impaired neural stem cell self-renewal, premature neuronal differentiation, and apoptosis resulting in degeneration of nearly the entire cortex. In vitro studies revealed that ATP6AP2 deficiency decreases V-ATPase membrane assembly and increases endosomal-lysosomal fusion. We conclude that ATP6AP2 is a key mediator of V-ATPase-dependent signaling and protein degradation in the developing human central nervous system.


Subject(s)
Central Nervous System/physiopathology , Neurodegenerative Diseases/diagnostic imaging , Neurodegenerative Diseases/genetics , Pluripotent Stem Cells/metabolism , Receptors, Cell Surface/genetics , Vacuolar Proton-Translocating ATPases/genetics , Adolescent , Alternative Splicing , Animals , Apoptosis , Brain/diagnostic imaging , Cell Death , Cell Differentiation , Cell Survival , Child, Preschool , Gene Deletion , Genetic Variation , HEK293 Cells , HeLa Cells , Humans , Lysosomes/metabolism , Male , Mice , Mice, Inbred C57BL , Neural Stem Cells/metabolism , Neurons/metabolism , Proton-Translocating ATPases/genetics , Proton-Translocating ATPases/physiology , Receptors, Cell Surface/physiology , Vacuolar Proton-Translocating ATPases/physiology
12.
Stem Cell Res ; 26: 8-16, 2018 01.
Article in English | MEDLINE | ID: mdl-29197744

ABSTRACT

MEN2A is a hereditary cancer-predisposing syndrome that affects patients with germline RET mutations. The effects of this oncogenic tyrosine kinase in the context of primitive stem cells are not known. In order to study these events, we generated a MEN2A induced Pluripotent Stem Cell (iPSC) line from a patient with RET mutation and an isogenic counterpart by CRISPR-Cas9 correction of the mutation. Whole exome sequencing of iPSC before and after CRISPR-Cas9 genome edition revealed no major exonic off target effect of the CRISPR correction. However, an integrative differential gene expression analysis of iPSC with oncogenic RETC634Y and its gene-corrected iPSC with RETY634C as well as RETwt iPSCs revealed activation of the Early Growth Response 1 (EGR1) transcriptional program in RET-mutated iPSC, a pathway shown to be involved in RET-induced oncogenesis. These data constitute the first proof of concept of the feasibility of the use of an iPSC and its genome-corrected counterpart to unravel the molecular mechanisms underlying the development of the hereditary MEN2A cancer predisposing syndrome.


Subject(s)
Clustered Regularly Interspaced Short Palindromic Repeats , Early Growth Response Protein 1/genetics , Induced Pluripotent Stem Cells/pathology , Multiple Endocrine Neoplasia Type 2a/genetics , Mutation , Proto-Oncogene Proteins c-ret/genetics , Transcriptome , Adult , Genome, Human , Humans , Induced Pluripotent Stem Cells/metabolism , Male , Multiple Endocrine Neoplasia Type 2a/pathology , Multiple Endocrine Neoplasia Type 2a/prevention & control , Proto-Oncogene Proteins c-ret/antagonists & inhibitors , Tumor Cells, Cultured
13.
Stem Cell Res ; 29: 56-59, 2018 05.
Article in English | MEDLINE | ID: mdl-29597128

ABSTRACT

Heterozygous non-synonymous (p.S142F) mutation in HNF1A leads to maturity-onset diabetes of the young (MODY) type 3, which is a subtype of dominant inherited young-onset non-autoimmune diabetes due to the defect of insulin secretion from pancreatic beta cells. We generated induced pluripotent stem cells (iPSCs) from a patient with HNF1A p.S142F mutation. Cells from this patient, which were reprogrammed by non-integrative viral transduction had normal karyotype, harboured the HNF1A p.S142F mutation, expressed pluripotency hallmarks.


Subject(s)
Diabetes Mellitus, Type 2/complications , Hepatocyte Nuclear Factor 1-alpha/metabolism , Induced Pluripotent Stem Cells/metabolism , Diabetes Mellitus, Type 2/pathology , Female , Humans , Male , Mutation
15.
Stem Cell Res ; 24: 135-138, 2017 10.
Article in English | MEDLINE | ID: mdl-29034880

ABSTRACT

BRCA1 germline mutation confers hereditary predisposition for breast and ovarian cancer. To understand the physiopathology of mammary and ovarian epithelial cancer transformation, and to identify early driver molecular events, we have generated an iPSC line from a patient carrying a germline exon 17 deletion in BRCA1 gene (BRAC1Ex17 iPSC) in a high-risk family context. Blood cells were reprogrammed used non-integrative virus of Sendaï. The BRCA1-deleted iPSC had normal karyotype, harboured a deletion in the exon 17 of the BRCA1 gene, expressed pluripotent hallmarks and had the differentiation capacity into the three germ layers.


Subject(s)
BRCA1 Protein/genetics , Exons/genetics , Induced Pluripotent Stem Cells/metabolism , Triple Negative Breast Neoplasms/genetics , BRCA1 Protein/metabolism , Cell Line , Female , Humans , Middle Aged , Sequence Deletion , Triple Negative Breast Neoplasms/metabolism , Triple Negative Breast Neoplasms/pathology
16.
Stem Cell Res ; 23: 178-181, 2017 08.
Article in English | MEDLINE | ID: mdl-28925365

ABSTRACT

Heterozygous activating mutation (p.Glu227Lys) in KCNJ11 leads to maturity-onset diabetes of the young (MODY) type 13, that is a subtype of dominant inherited young-onset non-autoimmune diabetes due to a primary defect in pancreatic beta cells. We generated induced pluripotent stem cells (iPSCs) from a patient with KCNJ11p.Glu227Lys mutation who developed MODY at 13years old. KCNJ11p.Glu227Lys-mutated cells that were reprogrammed by non-integrative viral transduction had normal karyotype, harboured the KCNJ11p.Glu227Lys mutation, expressed pluripotency hallmarks and had the differentiation capacity into the three germ layers.


Subject(s)
Cell Culture Techniques/methods , Diabetes Mellitus, Type 2/genetics , Diabetes Mellitus, Type 2/pathology , Induced Pluripotent Stem Cells/pathology , Mutation/genetics , Potassium Channels, Inwardly Rectifying/genetics , Animals , Base Sequence , Cell Shape , Humans , Male , Mice , Microsatellite Repeats/genetics , Middle Aged , Phenotype
17.
Exp Hematol ; 53: 48-58, 2017 09.
Article in English | MEDLINE | ID: mdl-28602946

ABSTRACT

We report here the first use of whole-genome sequencing (WGS) to examine the initial clonal dynamics in an unusual patient with chronic myeloid leukemia (CML), who presented in chronic phase (CP) with doubly marked BCR-ABL1+/JAK2V617F-mutant cells and, over a 9-year period, progressed into an accelerated phase (AP) and then terminal blast phase (BP). WGS revealed that the diagnostic cells also contained mutations in ASXL1, SEC23B, MAD1L1, and RREB1 as well as 12,000 additional uncommon DNA variants. WGS of endothelial cells generated from circulating precursors revealed many of these were shared with the CML clone. Surprisingly, WGS of induced pluripotent stem cells (iPSCs) derived from the AP cells revealed only six additional coding somatic mutations, despite retention by the hematopoietic progeny of the parental AP cell levels of BCR-ABL1 expression and sensitivity to imatinib and pimozide. Limited analysis of BP cells revealed independent subclonal progression to homozygosity of the MAD1L1 and RREB1 variants. MAD1L1 and SEC23B mutations were also identified in 2 of 101 cases of myeloproliferative neoplasms, but not in 42 healthy subjects. These findings challenge historic concepts of clonal evolution in CML.


Subject(s)
Janus Kinase 2/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Mutation , Cell Cycle Proteins/genetics , DNA-Binding Proteins/genetics , Genome-Wide Association Study , Humans , Induced Pluripotent Stem Cells/physiology , Male , Middle Aged , Nuclear Proteins/genetics , Transcription Factors/genetics
18.
Sci Rep ; 6: 27059, 2016 06 01.
Article in English | MEDLINE | ID: mdl-27245508

ABSTRACT

The pig is an emerging animal model, complementary to rodents for basic research and for biomedical and agronomical purposes. However despite the progress made on mouse and rat models to produce genuine pluripotent cells, it remains impossible to produce porcine pluripotent cell lines with germline transmission. Reprogramming of pig somatic cells using conventional integrative strategies remains also unsatisfactory. In the present study, we compared the outcome of both integrative and non-integrative reprogramming strategies on pluripotency and chromosome stability during pig somatic cell reprogramming. The porcine cell lines produced with integrative strategies express several pluripotency genes but they do not silence the integrated exogenes and present a high genomic instability upon passaging. In contrast, pig induced pluripotent-like stem cells produced with non-integrative reprogramming system (NI-iPSLCs) exhibit a normal karyotype after more than 12 months in culture and reactivate endogenous pluripotency markers. Despite the persistent expression of exogenous OCT4 and MYC, these cells can differentiate into derivatives expressing markers of the three embryonic germ layers and we propose that these NI-iPSLCs can be used as a model to bring new insights into the molecular factors controlling and maintaining pluripotency in the pig and other non-rodent mammalians.


Subject(s)
Cellular Reprogramming , Chromosomal Instability , Chromosomes, Mammalian/chemistry , Fibroblasts/metabolism , Induced Pluripotent Stem Cells/metabolism , Animals , Biomarkers/metabolism , Cell Cycle/genetics , Cell Differentiation , Cell Line , Fibroblasts/cytology , Gene Expression , Gene Expression Profiling , Genetic Vectors/chemistry , Genetic Vectors/metabolism , Induced Pluripotent Stem Cells/cytology , Karyotyping , Lentivirus/genetics , Lentivirus/metabolism , Octamer Transcription Factor-3/genetics , Octamer Transcription Factor-3/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , Swine
19.
PLoS One ; 11(3): e0149291, 2016.
Article in English | MEDLINE | ID: mdl-26938212

ABSTRACT

Hematopoiesis generated from human embryonic stem cells (ES) and induced pluripotent stem cells (iPS) are unprecedented resources for cell therapy. We compared hematopoietic differentiation potentials from ES and iPS cell lines originated from various donors and derived them using integrative and non-integrative vectors. Significant differences in differentiation toward hematopoietic lineage were observed among ES and iPS. The ability of engraftment of iPS or ES-derived cells in NOG mice varied among the lines with low levels of chimerism. iPS generated from ES cell-derived mesenchymal stem cells (MSC) reproduce a similar hematopoietic outcome compared to their parental ES cell line. We were not able to identify any specific hematopoietic transcription factors that allow to distinguish between good versus poor hematopoiesis in undifferentiated ES or iPS cell lines. There is a relatively unpredictable variation in hematopoietic differentiation between ES and iPS cell lines that could not be predicted based on phenotype or gene expression of the undifferentiated cells. These results demonstrate the influence of genetic background in variation of hematopoietic potential rather than the reprogramming process.


Subject(s)
Cellular Reprogramming/genetics , Embryonic Stem Cells/cytology , Genetic Heterogeneity , Graft Survival , Hematopoiesis/genetics , Induced Pluripotent Stem Cells/cytology , Animals , Biomarkers/metabolism , Cell Differentiation , Cell Line , Cell Lineage/genetics , Chimerism , Embryonic Stem Cells/metabolism , Embryonic Stem Cells/transplantation , Gene Expression , Genetic Vectors , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/transplantation , Lentivirus/genetics , Mice , Mice, Inbred NOD , Mice, SCID , Retroviridae/genetics , Tissue Donors , Transcription Factors/genetics , Transcription Factors/metabolism , Transplantation, Heterologous
20.
J Mol Endocrinol ; 34(1): 127-37, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15691883

ABSTRACT

Vascular endothelial-cadherin (VE-cadherin) is an endothelial cell-specific adhesion protein that is localised at cell-cell contacts. This molecule is an important determinant of vascular architecture and endothelial cell survival. In the adrenal cortex, steroidogenic and endothelial cells form a complex architecture. The adrenocorticotrophin hormone (ACTH) regulates gland homeostasis whose secretion is subjected to a negative feedback by adrenocorticosteroids. The aim of the present study was to determine whether VE-cadherin expression in the adrenal gland was regulated by hormonal challenge. We demonstrated that VE-cadherin protein levels were dramatically decreased (23.5+/-3.7%) by dexamethasone injections in the mouse and were restored by ACTH within 7 days (94.9+/-18.6%). Flow cytometry analysis of adrenal cells showed that the ratios of endothelial versus total adrenal cells were identical (35%) in dexamethasone- or ACTH-treated or untreated mice, suggesting that VE-cadherin expression could be regulated by ACTH. We demonstrate the existence of a transcriptional regulation of the VE-cadherin gene using transgenic mice carrying the chloramphenicol acetyl transferase gene under the control of the VE-cadherin promoter. Indeed, the promoter activity in the adrenals, but not in the lung or liver, was decreased in response to dexamethasone treatment (40+/-1.3%) and was partially restored after gland regeneration by ACTH injection (82+/-3%). In conclusion, our results show that transcription of a specific endothelial gene is controlled by the hypothalamo-pituitary axis and the data expand the knowledge regarding the role of ACTH in the regulation of the adrenal vascular network.


Subject(s)
Adrenal Glands/metabolism , Adrenocorticotropic Hormone/metabolism , Cadherins/genetics , Animals , Antigens, CD , Cadherins/biosynthesis , Endothelium/metabolism , Flow Cytometry , Mice , Promoter Regions, Genetic
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