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1.
Proc Natl Acad Sci U S A ; 120(19): e2220911120, 2023 05 09.
Article in English | MEDLINE | ID: mdl-37126681

ABSTRACT

Narcolepsy with cataplexy is a sleep disorder caused by deficiency in the hypothalamic neuropeptide hypocretin/orexin (HCRT), unanimously believed to result from autoimmune destruction of hypocretin-producing neurons. HCRT deficiency can also occur in secondary forms of narcolepsy and be only temporary, suggesting it can occur without irreversible neuronal loss. The recent discovery that narcolepsy patients also show loss of hypothalamic (corticotropin-releasing hormone) CRH-producing neurons suggests that other mechanisms than cell-specific autoimmune attack, are involved. Here, we identify the HCRT cell-colocalized neuropeptide QRFP as the best marker of HCRT neurons. We show that if HCRT neurons are ablated in mice, in addition to Hcrt, Qrfp transcript is also lost in the lateral hypothalamus, while in mice where only the Hcrt gene is inactivated Qrfp is unchanged. Similarly, postmortem hypothalamic tissues of narcolepsy patients show preserved QRFP expression, suggesting the neurons are present but fail to actively produce HCRT. We show that the promoter of the HCRT gene of patients exhibits hypermethylation at a methylation-sensitive and evolutionary-conserved PAX5:ETS1 transcription factor-binding site, suggesting the gene is subject to transcriptional silencing. We show also that in addition to HCRT, CRH and Dynorphin (PDYN) gene promoters, exhibit hypermethylation in the hypothalamus of patients. Altogether, we propose that HCRT, PDYN, and CRH are epigenetically silenced by a hypothalamic assault (inflammation) in narcolepsy patients, without concurrent cell death. Since methylation is reversible, our findings open the prospect of reversing or curing narcolepsy.


Subject(s)
Cataplexy , Narcolepsy , Neuropeptides , Mice , Animals , Orexins/metabolism , Cataplexy/genetics , Intracellular Signaling Peptides and Proteins/metabolism , Neuropeptides/metabolism , Narcolepsy/genetics , Hypothalamus/metabolism , Epigenesis, Genetic , Corticotropin-Releasing Hormone/genetics , Corticotropin-Releasing Hormone/metabolism
2.
Sleep Med Rev ; 60: 101546, 2021 12.
Article in English | MEDLINE | ID: mdl-34607185

ABSTRACT

Cataplexy is the pathognomonic and the most striking symptom of narcolepsy. It has originally been, and still is now, widely considered as an abnormal manifestation of rapid eye movement (REM) sleep during wakefulness due to the typical muscle atonia. The neurocircuits of cataplexy, originally confined to the brainstem as those of REM sleep atonia, now include the hypothalamus, dorsal raphe (DR), amygdala and frontal cortex, and its neurochemistry originally focused on catecholamines and acetylcholine now extend to hypocretin (HCRT) and other neuromodulators. Here, we review the neuroanatomy and neurochemistry of cataplexy and propose that cataplexy is a distinct brain state that, despite similarities with REM sleep, involves cataplexy-specific features.


Subject(s)
Cataplexy , Narcolepsy , Humans , Hypothalamus , Narcolepsy/diagnosis , Orexins , Sleep, REM/physiology , Wakefulness/physiology
4.
Nat Commun ; 11(1): 6034, 2020 11 27.
Article in English | MEDLINE | ID: mdl-33247179

ABSTRACT

Loss of muscle tone triggered by emotions is called cataplexy and is the pathognomonic symptom of narcolepsy, which is caused by hypocretin deficiency. Cataplexy is classically considered to be an abnormal manifestation of REM sleep and is treated by selective serotonin (5HT) reuptake inhibitors. Here we show that deleting the 5HT transporter in hypocretin knockout mice suppressed cataplexy while dramatically increasing REM sleep. Additionally, double knockout mice showed a significant deficit in the buildup of sleep need. Deleting one allele of the 5HT transporter in hypocretin knockout mice strongly increased EEG theta power during REM sleep and theta and gamma powers during wakefulness. Deleting hypocretin receptors in the dorsal raphe neurons of adult mice did not induce cataplexy but consolidated REM sleep. Our results indicate that cataplexy and REM sleep are regulated by different mechanisms and both states and sleep need are regulated by the hypocretinergic input into 5HT neurons.


Subject(s)
Cataplexy/physiopathology , Orexins/metabolism , Serotonin/metabolism , Sleep, REM/physiology , Animals , Cataplexy/genetics , Electroencephalography , Electromyography , Genotype , Mice, Knockout , Orexins/genetics , Serotonin/genetics , Theta Rhythm/physiology , Time Factors , Wakefulness/physiology
5.
Proc Natl Acad Sci U S A ; 116(34): 17061-17070, 2019 08 20.
Article in English | MEDLINE | ID: mdl-31375626

ABSTRACT

Hypocretin/orexin (HCRT) and melanin concentrating hormone (MCH) neuropeptides are exclusively produced by the lateral hypothalamus and play important roles in sleep, metabolism, reward, and motivation. Loss of HCRT (ligands or receptors) causes the sleep disorder narcolepsy with cataplexy in humans and in animal models. How these neuropeptides are produced and involved in diverse functions remain unknown. Here, we developed methods to sort and purify HCRT and MCH neurons from the mouse late embryonic hypothalamus. RNA sequencing revealed key factors of fate determination for HCRT (Peg3, Ahr1, Six6, Nr2f2, and Prrx1) and MCH (Lmx1, Gbx2, and Peg3) neurons. Loss of Peg3 in mice significantly reduces HCRT and MCH cell numbers, while knock-down of a Peg3 ortholog in zebrafish completely abolishes their expression, resulting in a 2-fold increase in sleep amount. We also found that loss of HCRT neurons in Hcrt-ataxin-3 mice results in a specific 50% decrease in another orexigenic neuropeptide, QRFP, that might explain the metabolic syndrome in narcolepsy. The transcriptome results were used to develop protocols for the production of HCRT and MCH neurons from induced pluripotent stem cells and ascorbic acid was found necessary for HCRT and BMP7 for MCH cell differentiation. Our results provide a platform to understand the development and expression of HCRT and MCH and their multiple functions in health and disease.


Subject(s)
Hypothalamic Hormones/metabolism , Hypothalamus/metabolism , Melanins/metabolism , Neurons/metabolism , Orexins/metabolism , Pituitary Hormones/metabolism , Animals , Hypothalamic Hormones/genetics , Hypothalamus/cytology , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Melanins/genetics , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Neurons/cytology , Orexins/genetics , Pituitary Hormones/genetics
6.
Lancet Neurol ; 14(3): 318-28, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25728441

ABSTRACT

The discovery of hypocretins (orexins) and their causal implication in narcolepsy is the most important advance in sleep research and sleep medicine since the discovery of rapid eye movement sleep. Narcolepsy with cataplexy is caused by hypocretin deficiency owing to destruction of most of the hypocretin-producing neurons in the hypothalamus. Ablation of hypocretin or hypocretin receptors also leads to narcolepsy phenotypes in animal models. Although the exact mechanism of hypocretin deficiency is unknown, evidence from the past 20 years strongly favours an immune-mediated or autoimmune attack, targeting specifically hypocretin neurons in genetically predisposed individuals. These neurons form an extensive network of projections throughout the brain and show activity linked to motivational behaviours. The hypothesis that a targeted immune-mediated or autoimmune attack causes the specific degeneration of hypocretin neurons arose mainly through the discovery of genetic associations, first with the HLA-DQB1*06:02 allele and then with the T-cell receptor α locus. Guided by these genetic findings and now awaiting experimental testing are models of the possible immune mechanisms by which a specific and localised brain cell population could become targeted by T-cell subsets. Great hopes for the identification of new targets for therapeutic intervention in narcolepsy also reside in the development of patient-derived induced pluripotent stem cell systems.


Subject(s)
Brain/physiopathology , Cataplexy/physiopathology , Intracellular Signaling Peptides and Proteins , Narcolepsy/physiopathology , Nerve Net/physiopathology , Neuropeptides , Animals , Brain/immunology , Cataplexy/genetics , Cataplexy/immunology , Humans , Intracellular Signaling Peptides and Proteins/genetics , Intracellular Signaling Peptides and Proteins/immunology , Narcolepsy/genetics , Narcolepsy/immunology , Nerve Net/immunology , Neuropeptides/genetics , Neuropeptides/immunology , Orexins
7.
Mol Biotechnol ; 54(3): 863-73, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23247991

ABSTRACT

The generation of human induced pluripotent stem cells (hiPSCs) from an individual patient provides a unique tool for disease modeling, drug discovery, and cell replacement therapies. Patient-specific pluripotent stem cells can be expanded in vitro and are thus suitable for genetic manipulations. To date, several genetic liver disorders have been modeled using patient-specific hiPSCs. Here, we present the generation of corrected hepatocyte-like cells (HLCs) from hiPSCs of a familial hypercholesterolemia (FH) patient with a homozygous mutation in the low-density lipoprotein receptor (LDLR) gene. We generated hiPSCs from a patient with FH with the mutated gene encoding a truncated non-functional receptor. In order to deliver normal LDLR to the defective cells, we used a plasmid vector carrying the normal receptor ORF to genetically transform the hiPSCs. The transformed cells were expanded and directed toward HLCs. Undifferentiated defective hiPSCs and HLCs differentiated from the defective hiPSCs did not have the ability to uptake labeled low-density lipoprotein (LDL) particles. The differentiated transformed hiPSCs showed LDL-uptake ability and the correction of disease phenotype as well as expressions of hepatocyte-specific markers. The functionality of differentiated cells was also confirmed by indo-cyanine green (ICG) uptake assay, PAS staining, inducible cyp450 activity, and oil red staining. These data suggest that hiPSC technology can be used for generation of disease-corrected, patient-specific HLCs with potential value for disease modeling and drug discovery as well as cell therapy applications in future.


Subject(s)
Cell Transplantation/methods , Genetic Therapy/methods , Hepatocytes/cytology , Hyperlipoproteinemia Type II/therapy , Induced Pluripotent Stem Cells/cytology , Adolescent , Biomarkers/analysis , Biomarkers/metabolism , Biotechnology , Female , Flow Cytometry , Humans , Immunohistochemistry , Karyotype , Mutation , Receptors, LDL/genetics , Transfection
8.
Stem Cells Dev ; 21(12): 2262-72, 2012 Aug 10.
Article in English | MEDLINE | ID: mdl-22145677

ABSTRACT

We describe a new, efficient protocol that involves the serial addition of noggin, basic fibroblast growth factor (bFGF), retinoic acid, and sonic hedgehog (Shh) for the differentiation of human induced pluripotent stem cells (hiPSC) to retinal pigmented epithelium (RPE) in a serum- and feeder-free adherent condition. hiPSC-RPE cells exhibited RPE morphology and specific molecular markers. Additionally, several hiPSC lines were generated from retinal-specific patients with Leber's congenital amaurosis, Usher syndrome, two patients with retinitis pigmentosa, and a patient with Leber's hereditary optic neuropathy. The RPE cells generated from these disease-specific hiPSCs expressed specific markers by the same RPE lineage-directed differentiation protocol. These findings indicate a new short-term, simple, and efficient protocol for differentiation of hiPSCs to RPE cells. Such specific retinal disease-specific hiPSCs offer an unprecedented opportunity to recapitulate normal and pathologic formation of human retinal cells in vitro, thereby enabling pharmaceutical screening, and potentially autologous cell replacement therapies for retinal diseases.


Subject(s)
Cell Differentiation , Induced Pluripotent Stem Cells/physiology , Retinal Diseases/pathology , Retinal Pigment Epithelium/pathology , Adolescent , Adult , Antigens, Differentiation/genetics , Antigens, Differentiation/metabolism , Cell Culture Techniques , Cell Shape , Cells, Cultured , Child , Female , Gene Expression , Humans , Induced Pluripotent Stem Cells/metabolism , Karyotype , Male , Middle Aged , Phenotype , Real-Time Polymerase Chain Reaction , Retinal Pigment Epithelium/metabolism
9.
Stem Cells Dev ; 20(11): 1911-23, 2011 Nov.
Article in English | MEDLINE | ID: mdl-21198400

ABSTRACT

Traditionally, undifferentiated pluripotent human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) have been expanded as monolayer colonies in adhesion culture, both in the presence or absence of feeder cells. However, the use of pluripotent stem cells poses the need to scale-up current culture methods. Herein, we present the cultivation of 2 hESC lines (Royan H5 and Royan H6) and 2 hiPSC lines (hiPSC1 and hiPSC4) as carrier-free suspension aggregates for an extended period of time. The cells proliferated over multiple passages kept a stable karyotype, which successfully maintained an undifferentiated state and pluripotency, as determined by marker expressions in addition to in vitro spontaneous and directed differentiation. Additionally, these cells can be easily frozen and thawed without losing their proliferation, karyotype stability, and developmental potential. Transcriptome analysis of the 3 lines revealed that the adherent culture condition was nearly identical to the suspension culture in Royan H5 and hiPSC1, but not in Royan H6. It remains unclear whether this observation at the transcript level is biologically significant. In comparison with recent reports, our study presents a low-cost procedure for long-term suspension expansion of hESCs and hiPSCs with the capability of freeze/thawing, karyotype stability, and pluripotency. Our results will pave the way for scaled up expansion and controlled differentiation of hESCs and hiPSCs needed for cell therapy, research, and industrial applications in a bioreactor culture system.


Subject(s)
Embryonic Stem Cells/cytology , Induced Pluripotent Stem Cells/cytology , Antigens, Differentiation/metabolism , Apoptosis , Cell Aggregation , Cell Culture Techniques , Cell Differentiation , Cell Shape , Cells, Cultured , Cryopreservation , Culture Media, Conditioned , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/physiology , Fibroblast Growth Factor 2/pharmacology , Fibroblast Growth Factor 2/physiology , Gene Expression Profiling , Humans , Induced Pluripotent Stem Cells/drug effects , Induced Pluripotent Stem Cells/physiology , Karyotype , Peptide Fragments/pharmacology , rho-Associated Kinases/antagonists & inhibitors
10.
Arch Iran Med ; 13(6): 573-5, 2010 Nov.
Article in English | MEDLINE | ID: mdl-21039019
11.
Stem Cell Rev Rep ; 6(4): 622-32, 2010 Dec.
Article in English | MEDLINE | ID: mdl-20821352

ABSTRACT

The availability of disease-specific induced pluripotent stem cells (iPSCs) offers a unique opportunity for studying and modeling the effects of specific gene defects on human liver development in vitro and for testing small molecules or other potential therapies for relevant liver disorders. Here we report, for the first time, the derivation of iPSCs by the retroviral transduction of Yamanaka's factors in serum and feeder-free culture conditions from liver-specific patients with tyrosinemia, glycogen storage disease, progressive familial hereditary cholestasis, and two siblings with Crigler-Najjar syndrome. Furthermore, they were differentiated into functional hepatocyte-like cells efficiently. These iPSCs possessed properties of human embryonic stem cells (hESCs) and were successfully differentiated into three lineages that resembled hESC morphology, passaging, surface and pluripotency markers, normal karyotype, DNA methylation, and differentiation. The hepatic lineage-directed differentiation showed that the iPSC-derived hepatic cells expressed hepatocyte-specific markers. Their functionality was confirmed by glycogen and lipid storage activity, secretion of albumin, alpha-fetoprotein, and urea, CYP450 metabolic activity, as well as LDL and indocyanin green uptake. Our results provide proof of principal that human liver-disease specific iPSCs present an exciting potential venue toward cell-based therapeutics, drug metabolism, human liver development and disease models for liver failure disorders.


Subject(s)
Cell Differentiation/physiology , Hepatocytes/cytology , Induced Pluripotent Stem Cells/cytology , Liver Diseases/pathology , Adolescent , Adult , Alkaline Phosphatase/metabolism , Cells, Cultured , Child , Female , Fibroblasts/cytology , Flow Cytometry , Fluorescent Antibody Technique , Humans , Induced Pluripotent Stem Cells/pathology , Karyotyping , Lipoproteins, LDL/metabolism , Liver Diseases/metabolism , Liver Diseases/physiopathology , Male , Young Adult
12.
Stem Cell Rev Rep ; 6(2): 297-306, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20180049

ABSTRACT

The recent generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an invaluable resource for drug or toxicology screening, medical research, and patient-specific cell therapy. However, there are currently a number of obstacles including virus integration and the genetic alteration of iPSCs that will need to be overcome before these cells may be considered safe for clinical applications. Here, we highlight the potential and challenges of iPSC research and review advances in reprogramming methods that have rapidly moved the field closer to realizing the goal of generating safe iPSCs for transplantation.


Subject(s)
Cell- and Tissue-Based Therapy/methods , Induced Pluripotent Stem Cells/cytology , Humans
13.
In Vitro Cell Dev Biol Anim ; 46(3-4): 395-402, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20177993

ABSTRACT

In this study, we focused on the derivation of human embryonic stem cell (hESC) from preimplantation genetic screening (PGS)-analyzed and preimplantation genetic diagnosis (PGD)-analyzed embryos. Out of 62 fresh PGD/PGS-analyzed embryos, 22 embryos reached the blastocyst stage. From 12 outgrowth blastocysts, we derived four hESC lines onto a feeder layer. Surprisingly, karyotype analysis showed that hESC lines derived from aneuploid embryos had diploid female karyotype. One hESC line was found to carry a balanced Robertsonian translocation. All the cell lines showed hESC markers and had the pluripotent ability to differentiate into derivatives of the three embryonic germ layers. The established lines had clonal propagation with 22-31% efficiency in the presence of ROCK inhibitor. These results further indicate that hESC lines can be derived from PGD/PGS-analyzed embryos that are destined to be discarded and can serve as an alternative source for normal euploid lines.


Subject(s)
Cell Culture Techniques/methods , Embryo, Mammalian/cytology , Embryonic Stem Cells/cytology , Preimplantation Diagnosis/methods , Animals , Blastomeres/cytology , Cell Differentiation , Cell Line , Colony-Forming Units Assay , Humans , In Situ Hybridization, Fluorescence , Karyotyping , Mice
14.
Methods Mol Biol ; 584: 425-43, 2010.
Article in English | MEDLINE | ID: mdl-19907991

ABSTRACT

The recent discovery of genomic reprogramming of human somatic cells to an embryonic stem (ES) cell-like pluripotent state provides a unique opportunity for stem cell research. The reprogrammed cells, named as induced pluripotent stem (iPS) cells, possess many of the properties of ES cells and represent one of the most promising sources of patient-specific cells for use in disease model, development of pharmacology and toxicology, screening teratogens, and regenerative medicine. Here we describe the detailed methods for the generation of undifferentiated human iPS (hiPS) cells in feeder layer- and serum-free conditions. This system eliminates direct contact of stem cells with MEFs and reduces use of unknown serum factors that may have undesired activities and enables consistency in large-scale and long-term expansion of undifferentiated hiPS cells. Our findings greatly simplify the method for induction of pluripotency and bring it one step closer to clinical applications. Moreover, the established hiPS cells showed chromosomal stability during long-term culture.


Subject(s)
Pluripotent Stem Cells/cytology , Adult Stem Cells/cytology , Adult Stem Cells/metabolism , Animals , Biomarkers/metabolism , Cell Culture Techniques/methods , Cell Dedifferentiation , Chromosomal Instability , Coculture Techniques , Cryopreservation/methods , Culture Media, Serum-Free , Fibroblasts/cytology , Genetic Vectors , Humans , Immunohistochemistry , Karyotyping , Mice , Pluripotent Stem Cells/metabolism , Skin/cytology , Transfection
15.
Biochem Biophys Res Commun ; 391(1): 329-34, 2010 Jan 01.
Article in English | MEDLINE | ID: mdl-19912985

ABSTRACT

Bombay phenotype is one of the rare phenotypes in the ABO blood group system that fails to express ABH antigens on red blood cells. Nonsense or missense mutations in fucosyltransfrase1 (FUT1) and fucosyltransfrase2 (FUT2) genes are known to create this phenotype. This blood group is compatible with all other blood groups as a donor, as it does not express the H antigen on the red blood cells. In this study, we describe the establishment of human induced pluripotent stem cells (iPSCs) from the dermal fibroblasts of a Bombay blood-type individual by the ectopic expression of established transcription factors Klf4, Oct4, Sox2, and c-Myc. Sequence analyses of fibroblasts and iPSCs revealed a nonsense mutation 826C to T (276 Gln to Ter) in the FUT1 gene and a missense mutation 739G to A (247 Gly to Ser) in the FUT2 gene in the Bombay phenotype under study. The established iPSCs resemble human embryonic stem cells in morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, DNA methylation of critical pluripotency genes, and in-vitro differentiation. The directed differentiation of the iPSCs into hematopoietic lineage cells displayed increased expression of the hematopoietic lineage markers such as CD34, CD133, RUNX1, KDR, alpha-globulin, and gamma-globulin. Such specific stem cells provide an unprecedented opportunity to produce a universal blood group donor, in-vitro, thus enabling cellular replacement therapies, once the safety issue is resolved.


Subject(s)
ABO Blood-Group System , Cell Line , Erythrocytes/cytology , Hematopoiesis , Pluripotent Stem Cells/cytology , Amino Acid Sequence , Base Sequence , Blood Donors , Fibroblasts/cytology , Fibroblasts/metabolism , Fucosyltransferases/genetics , Gene Expression , Humans , Kruppel-Like Factor 4 , Mutation, Missense , Phenotype , Pluripotent Stem Cells/metabolism , Transcription Factors/genetics , Galactoside 2-alpha-L-fucosyltransferase
16.
Int J Dev Biol ; 54(5): 877-86, 2010.
Article in English | MEDLINE | ID: mdl-19876814

ABSTRACT

Although human induced pluripotent stem cells (hiPSCs) hold great promise as a source of differentiated cells for vast therapeutic implications, many obstacles still need to be surmounted before this can become a reality. One obstacle, a robust feeder- and serum-free system to generate and expand hiPSCs in culture is still unavailable. Here, for the first time, we describe a novel establishment and maintenance culture technique that uses human dermal fibroblasts to generate hiPSCs by introducing four factors, Klf4, Oct4, Sox2, and c-Myc under serum- and feeder-independent conditions. We have used a serum replacement product, conditioned medium (CM), or feeder-free medium (FFM) supplemented with high elevated basic-fibroblast growth factor in the absence or presence of Matrigel. Our FFM system in the presence of Matrigel enhanced the efficiency of alkaline phosphatase-positive colonies at a frequency at least 10-fold greater than the conventional method on feeder cells. The established hiPSCs are similar to human embryonic stem cells in many aspects including morphology, passaging, surface and pluripotency markers, normal karyotype, gene expression, ultrastructure, and in vitro differentiation. Such hiPSCs could be useful particularly in the context of in vitro disease modeling, pharmaceutical screening and in cellular replacement therapies once the safety issues have been overcome.


Subject(s)
Cell Culture Techniques/methods , Cell Differentiation/genetics , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/metabolism , Adult , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Culture Media, Serum-Free/pharmacology , Fluorescent Antibody Technique , Gene Expression/drug effects , Humans , Induced Pluripotent Stem Cells/ultrastructure , Karyotyping , Kruppel-Like Factor 4 , Microscopy, Electron, Scanning , Microscopy, Electron, Transmission , Reverse Transcriptase Polymerase Chain Reaction
17.
Stem Cell Rev Rep ; 6(1): 96-107, 2010 Mar.
Article in English | MEDLINE | ID: mdl-20012714

ABSTRACT

Optimization and development of better defined culture methods for human embryonic and induced pluripotent stem cells (hESCs and hiPSCs) will provide an invaluable contribution to the field of regenerative medicine. However, one problem is the vulnerability of hESCs and hiPSCs to apoptosis that causes a low plating efficiency upon passaging. Herein, we have developed a novel hESCs and hiPSCs culture technique that uses ROCK inhibitor (ROCKi) Y-27632 (10 microM) in Matrigel-coated dishes in both serum- and feeder-free culture conditions. This increases plating efficiency during enzymatic and mechanical passaging as compared to its presence solely in culture medium. Under these conditions, hESCs (three lines) and hiPSCs (two lines) retain their typical morphology, a stable karyotype, express pluripotency markers and have the potential to differentiate into derivatives of all three germ layers after long-term culture. Real-time RT-PCR analysis of stemness-related integrins (alphaV, alpha6, and beta1) has demonstrated that their expression increases in the presence of ROCKi. Similar plating efficiencies have been obtained in both hESCs and hiPSCs with a lower concentration of Y-27632 (800 nM) and another ROCKi (HA-1077/Fasudil), thus ruling out the non-specific effects of Y-27632. These results show that addition of ROCKi in the extracellular matrix can increase the plating efficiency of hESCs and hiPSCs during passaging of clusters. This is due not only to an anti-apoptotic effect, but also to an increase in the ECM-cells interaction. Therefore, we believe this method will be useful for both current and future applications of these pluripotent stem cells.


Subject(s)
Cell Proliferation/drug effects , Collagen/metabolism , Embryonic Stem Cells/drug effects , Extracellular Matrix/metabolism , Induced Pluripotent Stem Cells/drug effects , Laminin/metabolism , Protein Kinase Inhibitors/pharmacology , Proteoglycans/metabolism , rho-Associated Kinases/antagonists & inhibitors , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , Amides/pharmacology , Apoptosis/drug effects , Biomarkers/metabolism , Cell Differentiation/drug effects , Cell Line , Cell Shape/drug effects , Drug Carriers , Drug Combinations , Embryonic Stem Cells/enzymology , Gene Expression Regulation, Developmental/drug effects , Humans , Induced Pluripotent Stem Cells/enzymology , Integrin alpha6/genetics , Integrin alphaV/genetics , Integrin beta1/genetics , Karyotyping , Pyridines/pharmacology , RNA, Messenger/metabolism , rho-Associated Kinases/metabolism
18.
Hum Reprod ; 24(10): 2468-76, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19602515

ABSTRACT

BACKGROUND: An essential prerequisite for the future widespread application of human induced pluripotent (hiPSCs) and embryonic stem cells (hESCs) is the development of efficient cryopreservation methods to facilitate their storage and transportation. METHODS: We developed a simple and effective freezing/thawing method of single dissociated hESCs and hiPSCs in a feeder-free culture in the presence of Rho-associated kinase (ROCK) inhibitor Y-27632. RESULTS: Exposure to ROCK inhibitor Y-27632 in freezing solution alone does not significantly enhance the post-thaw survival rate of single dissociated hESCs and hiPSCs. However, when ROCK inhibitor was added to both pre- and post-thaw culture media, there was an enhancement in the survival rate, which further increased when ROCK inhibitor was added to Matrigel as well. Under these treatments, hESCs and hiPSCs retained typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture. CONCLUSIONS: This method is an effective cryopreservation procedure for single dissociated hESCs in feeder-free culture, which is also applicable for single dissociated hiPSCs using a ROCK inhibitor. The cloning efficiency of hiPSCs and hESCs improves when ROCK inhibitor is added both in Matrigel and in medium in comparison with conventional addition to medium. Therefore, we believe this method would be useful for current and future applications of the pluripotent stem cells.


Subject(s)
Cryopreservation/methods , Embryonic Stem Cells/cytology , Pluripotent Stem Cells/cytology , Cell Culture Techniques , Cell Differentiation , Cell Survival , Cells, Cultured , Collagen , Cryoprotective Agents , Culture Media , Drug Combinations , Embryonic Stem Cells/metabolism , Genetic Markers , Humans , Laminin , Pluripotent Stem Cells/metabolism , Proteoglycans
19.
J Invertebr Pathol ; 102(2): 101-9, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19631215

ABSTRACT

The characterization of the strains containing Coleopteran-specific and also putative novel cry genes in Iranian native Bacillus thuringiensis collection is presented. Characterization was based on PCR analysis using 31 general and specific primers for cry1B, cry1I, cry3A, cry3B, cry3C, cry7A, cry8A, cry8B, cry8C, cry14, cry18, cry26, cry28, cry34 and cry35 genes, protein band patterns as well as their insecticidal activity on Xanthogaleruca luteola Mull. larvae. Forty six isolates (65.7%) contained minimum one Coleopteran-active cry gene. Based on universal primers, strains containing cry18 and cry26 genes were the most abundant and represent 27.1% and 24% of the isolates, respectively, whereas cry14, cry3, cry28, cry34, cry35, cry7, cry8 genes were less abundant, found in 14.2, 12.5, 10, 7, 7 and 5.6% of the strains, respectively. Based on specific primers, isolates containing cry1I were the most abundant (48.5%). Two strains containing Coleopteran-active cry genes showed higher activity against X. luteola larvae than B. thuringiensis subsp. morrisoni pathovar tenebrionis. Thirty isolates, when assayed for cry1C, cry5, cry6, cry8b, cry9, cry10, cry11, cry18, cry24 and cry35 genes, showed unexpected size bands. Cloning and sequencing of the amplicons allowed both the identification of known cry genes and the detection of putative novel cry1C sequences.


Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Coleoptera/microbiology , Genes, Bacterial/genetics , Genes, Insect/genetics , Animals , Cloning, Molecular , Coleoptera/genetics , Electrophoresis, Agar Gel/veterinary , Iran , Polymerase Chain Reaction/methods , Polymerase Chain Reaction/veterinary
20.
Can J Microbiol ; 54(10): 812-22, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18923549

ABSTRACT

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nematodes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 x 10(8) CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 x 10(8) CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.


Subject(s)
Bacillus thuringiensis/genetics , Bacterial Proteins/genetics , Endotoxins/genetics , Hemolysin Proteins/genetics , Nematoda/microbiology , Pest Control, Biological/methods , Plants/parasitology , Animals , Bacillus thuringiensis/isolation & purification , Bacillus thuringiensis/metabolism , Bacillus thuringiensis Toxins , Bacterial Proteins/metabolism , Bacterial Proteins/toxicity , Endotoxins/metabolism , Endotoxins/toxicity , Hemolysin Proteins/metabolism , Hemolysin Proteins/toxicity , Iran , Molecular Sequence Data , Nematoda/isolation & purification , Polymerase Chain Reaction , Soil/parasitology , Soil Microbiology
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