scholarly journals Generation of an isogenic set of HD model hESC lines v1 (protocols.io.bursnv6e)

protocols.io ◽  
2021 ◽  
Author(s):  
Albert Ruzo ◽  
Gist F. ◽  
Jakob J. ◽  
Szilvia Galgoczi ◽  
Lauren J. ◽  
...  
Keyword(s):  
Hesc Lines

scholarly journals MFN2 Deficiency Impairs Mitochondrial Transport and Downregulates Motor Protein Expression in Human Spinal Motor Neurons

2021 ◽  
Vol 14 ◽  
Author(s):  
Yongchao Mou ◽  
Joshua Dein ◽  
Zhenyu Chen ◽  
Mrunali Jagdale ◽  
Xue-Jun Li
Keyword(s):  
Protein Expression ◽  
Motor Neurons ◽  
Axonal Degeneration ◽  
Axonal Neuropathy ◽  
Motor Protein ◽  
Mitochondrial Morphology ◽  
Mitochondrial Transport ◽  
Spinal Motor Neurons ◽  
Spinal Motor ◽  
Hesc Lines

Charcot-Marie-Tooth (CMT) disease is one of the most common genetically inherited neurological disorders and CMT type 2A (CMT 2A) is caused by dominant mutations in the mitofusin-2 (MFN2) gene. MFN2 is located in the outer mitochondrial membrane and is a mediator of mitochondrial fusion, with an essential role in maintaining normal neuronal functions. Although loss of MFN2 induces axonal neuropathy, the detailed mechanism by which MFN2 deficiency results in axonal degeneration of human spinal motor neurons remains largely unknown. In this study, we generated MFN2-knockdown human embryonic stem cell (hESC) lines using lentivirus expressing MFN2 short hairpin RNA (shRNA). Using these hESC lines, we found that MFN2 loss did not affect spinal motor neuron differentiation from hESCs but resulted in mitochondrial fragmentation and dysfunction as determined by live-cell imaging. Notably, MFN2-knockodwn spinal motor neurons exhibited CMT2A disease-related phenotypes, including extensive perikaryal inclusions of phosphorylated neurofilament heavy chain (pNfH), frequent axonal swellings, and increased pNfH levels in long-term cultures. Importantly, MFN2 deficit impaired anterograde and retrograde mitochondrial transport within axons, and reduced the mRNA and protein levels of kinesin and dynein, indicating the interfered motor protein expression induced by MFN2 deficiency. Our results reveal that MFN2 knockdown induced axonal degeneration of spinal motor neurons and defects in mitochondrial morphology and function. The impaired mitochondrial transport in MFN2-knockdown spinal motor neurons is mediated, at least partially, by the altered motor proteins, providing potential therapeutic targets for rescuing axonal degeneration of spinal motor neurons in CMT2A disease.

scholarly journals Blockade of STAT3 Causes Severe In Vitro and In Vivo Maturation Defects in Intestinal Organoids Derived from Human Embryonic Stem Cells

2019 ◽  
Vol 8 (7) ◽  
pp. 976 ◽  
Author(s):  
Kwang Bo Jung ◽  
Ohman Kwon ◽  
Mi-Ok Lee ◽  
Hana Lee ◽  
Ye Seul Son ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Embryonic Stem Cell ◽  
In Vitro Maturation ◽  
Embryonic Stem ◽  
Generating Functional ◽  
Intestinal Maturation ◽  
Intestinal Organoids ◽  
Hesc Lines

Human intestinal organoids (hIOs), which resemble the human intestine structurally and physiologically, have emerged as a new modality for the study of the molecular and cellular biology of the intestine in vitro. We recently developed an in vitro maturation technique for generating functional hIOs from human pluripotent stem cells (hPSCs). Here, we investigated the function of STAT3 for inducing in vitro maturation of hIOs. This was accompanied by the tyrosine phosphorylation of STAT3, whereas treatment with pharmacological inhibitors of STAT3 suppressed the phosphorylation of STAT3 and the expression of intestinal maturation markers. We generated and characterized STAT3 knockout (KO) human embryonic stem cell (hESC) lines using CRISPR/Cas9-mediated gene editing. We found that STAT3 KO does not affect the differentiation of hESCs into hIOs but rather affects the in vitro maturation of hIOs. STAT3 KO hIOs displayed immature morphologies with decreased size and reduced budding in hIOs even after in vitro maturation. STAT3 KO hIOs showed markedly different profiles from hIOs matured in vitro and human small intestine. Additionally, STAT3 KO hIOs failed to maintain upon in vivo transplantation. This study reveals a core signaling pathway consisting of STAT3 controlling the in vitro maturation of hIOs derived from hPSCs.

Derivation of human embryonic stem cell lines after blastocyst microsurgery

2010 ◽  
Vol 88 (3) ◽  
pp. 479-490 ◽  
Author(s):  
Guoliang Meng ◽  
Shiying Liu ◽  
Xiangyun Li ◽  
Roman Krawetz ◽  
Derrick E. Rancourt
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Lines ◽  
Clinical Medicine ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Hesc Lines

Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of the blastocyst. Because of their ability to differentiate into a variety of cell types, human embryonic stem cells (hESCs) provide an unlimited source of cells for clinical medicine and have begun to be used in clinical trials. Presently, although several hundred hESC lines are available in the word, only few have been widely used in basic and applied research. More and more hESC lines with differing genetic backgrounds are required for establishing a bank of hESCs. Here, we report the first Canadian hESC lines to be generated from cryopreserved embryos and we discuss how we navigated through the Canadian regulatory process. The cryopreserved human zygotes used in this study were cultured to the blastocyst stage, and used to isolate ICM via microsurgery. Unlike previous microsurgery methods, which use specialized glass or steel needles, our method conveniently uses syringe needles for the isolation of ICM and subsequent hESC lines. ICM were cultured on MEF feeders in medium containing FBS or serum replacer (SR). Resulting outgrowths were isolated, cut into several cell clumps, and transferred onto fresh feeders. After more than 30 passages, the two hESC lines established using this method exhibited normal morphology, karyotype, and growth rate. Moreover, they stained positively for a variety of pluripotency markers and could be differentiated both in vitro and in vivo. Both cell lines could be maintained under a variety of culture conditions, including xeno-free conditions we have previously described. We suggest that this microsurgical approach may be conducive to deriving xeno-free hESC lines when outgrown on xeno-free human foreskin fibroblast feeders.

scholarly journals CRISPR/Cas9 mediated generation of human ARID1B heterozygous knockout hESC lines to model Coffin-Siris syndrome

2020 ◽  
Vol 47 ◽  
pp. 101889
Author(s):  
Tom Boerstler ◽  
Holger Wend ◽  
Mandy Krumbiegel ◽  
Atria Kavyanifar ◽  
André Reis ◽  
...  
Keyword(s):  
Hesc Lines

scholarly journals Enhanced Immunological Tolerance by HLA-G1 from Neural Progenitor Cells (NPCs) Derived from Human Embryonic Stem Cells (hESCs)

2017 ◽  
Vol 44 (4) ◽  
pp. 1435-1444 ◽  
Author(s):  
Hong-Xi Zhao ◽  
Feng Jiang ◽  
Ya-Jing Zhu ◽  
Li Wang ◽  
Ke Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Human Embryonic Stem Cells ◽  
Immune Tolerance ◽  
Neural Progenitor Cells ◽  
Embryonic Stem ◽  
Immunological Tolerance ◽  
Self Renewal ◽  
Hesc Lines

Background: Despite the great potential of utilizing human embryonic stem cells (hESCs)-derived cells as cell source for transplantation, these cells were often rejected during engraftment by the immune system due to adaptive immune response. Methods: We first evaluated HLA-G expression level in both hESCs and differentiated progenitor cells. After that, we generated modified hESC lines that over-express HLA-G1 using lentiviral infection with the construct contains both HLA-G1 and GFP tag. The lentivirus was first produced by co-transfecting HLA-G1 expressing lentiviral vector together with packaging vectors into packaging cell line 293T. Then the produced virus was used for the infection of selected hESC lines. We characterized the generated cell lines phenotype, including pluripotency and self-renewal abilities, as well as immune tolerance ability by mixed lymphocyte reaction (MLR) and cytotoxicity assays. Results: Although the hESCs do not express high levels of HLA-G1, over-expression of HLA-G1 in hESCs still retains their stem cell characteristics as determined by retaining the expression levels of OCT4 and SOX2, two critical transcriptional factors for stem cell function. Furthermore, the HLA-G1 overexpressing hESCs retain the self-renewal and pluripotency characteristics of stem cells, which can differentiate into different types of cells, including pigment cells, smooth muscle cells, epithelia-like cells, and NPCs. After differentiation, the differentiated cells including NPCs retain the high levels of HLA-G1 protein. In comparison with conventional NPCs, these HLA-G1 positive NPCs have enhanced immune tolerance ability. Conclusions: Ectopic expression of HLA-G1, a non-classical major histocompatibility complex class I (MHC I) antigen that was originally discovered involving in engraftment tolerance during pregnancy, can enhance the immunological tolerance in differentiated neural progenitor cells (NPCs). Our study shows that stably overexpressing HLA-G1 in hESCs might be a feasible strategy for enhancing the engraftment of NPCs during transplantation.

scholarly journals Expression profiles of protein tyrosine kinase genes in human embryonic stem cells

Reproduction ◽  
2008 ◽  
Vol 136 (4) ◽  
pp. 423-432 ◽  
Author(s):  
Mi-Young Son ◽  
Janghwan Kim ◽  
Hyo-Won Han ◽  
Sun-Mi Woo ◽  
Yee Sook Cho ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Signaling Pathways ◽  
Human Embryonic Stem Cells ◽  
Tyrosine Kinases ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Degenerate Primers ◽  
Protein Tyrosine ◽  
Hesc Lines

Complex signaling pathways operate in human embryonic stem cells (hESCs) and are coordinated to maintain self-renewal and stem cell characteristics in them. Protein tyrosine kinases (PTKs) participate in diverse signaling pathways in various types of cells. Because of their functions as key molecules in various cellular processes, PTKs are anticipated to have important roles also in hESCs. In this study, we investigated the roles of PTKs in undifferentiated and differentiated hESCs. To establish comprehensive PTK expression profiles in hESCs, we performed reverse transcriptase PCR using degenerate primers according to the conserved catalytic PTK motifs in both undifferentiated and differentiated hESCs. Here, we identified 42 different kinases in two hESC lines, including 5 non-receptor tyrosine kinases (RTKs), 24 RTKs, and 13 dual and other kinases, and compared the protein kinase expression profiles of hESCs and retinoic acid-treated hESCs. Significantly, up- and downregulated kinases in undifferentiated hESCs were confirmed by real-time PCR and western blotting. MAP3K3, ERBB2, FGFR4, and EPHB2 were predominantly upregulated, while CSF1R, TYRO3, SRC, and GSK3A were consistently downregulated in two hESC lines. Western blot analysis showed that the transcriptional levels of these kinases were consistent with the translational levels. The obstruction of upregulated kinases’ activities using specific inhibitors disturbed the undifferentiated status and induced the differentiation of hESCs. Our results support the dynamic expression of PTKs during hESC maintenance and suggest that specific PTKs that are consistently up- and downregulated play important roles in the maintenance of stemness and the direction of differentiation of hESCs.

scholarly journals Celebrating 10 Years of hESC Lines: An Interview with James Thomson

Stem Cells ◽  
2008 ◽  
Vol 26 (11) ◽  
pp. 2747-2748
Author(s):  
Miodrag Stojković ◽  
Susan Rainey Daher
Keyword(s):  
James Thomson ◽  
Hesc Lines

scholarly journals Heterozygous APC germline mutations impart predisposition to colorectal cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Livia Preisler ◽  
Aline Habib ◽  
Guy Shapira ◽  
Liron Kuznitsov-Yanovsky ◽  
Yoav Mayshar ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Rna Sequencing ◽  
Disease Severity ◽  
Germline Mutations ◽  
Apc Gene ◽  
Adenomatous Polyposis ◽  
Loss Of Function ◽  
Tumorigenic Transformation ◽  
Hesc Lines ◽  
Apc Mutation

AbstractFamilial adenomatous polyposis (FAP) is an inherited syndrome caused by a heterozygous adenomatous polyposis coli (APC) germline mutation, associated with a profound lifetime risk for colorectal cancer. While it is well accepted that tumorigenic transformation is initiated following acquisition of a second mutation and loss of function of the APC gene, the role of heterozygous APC mutation in this process is yet to be discovered. This work aimed to explore whether a heterozygous APC mutation induces molecular defects underlying tumorigenic transformation and how different APC germline mutations predict disease severity. Three FAP-human embryonic stem cell lines (FAP1/2/3-hESC lines) carrying germline mutations at different locations of the APC gene, and two control hESC lines free of the APC mutation, were differentiated into colon organoids and analyzed by immunohistochemistry and RNA sequencing. In addition, data regarding the genotype and clinical phenotype of the embryo donor parents were collected from medical records. FAP-hESCs carrying a complete loss-of-function of a single APC allele (FAP3) generated complex and molecularly mature colon organoids, which were similar to controls. In contrast, FAP-hESCs carrying APC truncation mutations (FAP1 and FAP2) generated only few cyst-like structures and cell aggregates of various shape, occasionally with luminal parts, which aligned with their failure to upregulate critical differentiation genes early in the process, as shown by RNA sequencing. Abnormal disease phenotype was shown also in non-pathological colon of FAP patients by the randomly distribution of proliferating cells throughout the crypts, compared to their focused localization in the lower part of the crypt in healthy/non-FAP patients. Genotype/phenotype analysis revealed correlations between the colon organoid maturation potential and FAP severity in the carrier parents. In conclusion, this study suggest that a single truncated APC allele is sufficient to initiate early molecular tumorigenic activity. In addition, the results hint that patient-specific hESC-derived colon organoids can probably predict disease severity among FAP patients.

scholarly journals Interactome comparison of human embryonic stem cell lines with the inner cell mass and trophectoderm

2018 ◽  
Author(s):  
Adam Stevens ◽  
Helen Smith ◽  
Terence Garner ◽  
Ben Minogue ◽  
Sharon Sneddon ◽  
...  
Keyword(s):  
Human Embryonic Stem Cell ◽  
Transcriptional Activity ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Specific Gene ◽  
Transcriptomic Data ◽  
Network Properties ◽  
Stem Cell Lines ◽  
Hesc Lines

AbstractHuman embryonic stem cells (hESCs) derived from the pluripotent Inner cell mass (ICM) of the blastocyst are fundamental tools for understanding human development, yet are not identical to their tissue of origin. To investigate this divergence we compared the transcriptomes of genetically paired ICM and trophectoderm (TE) samples with three hESC lines: MAN1, HUES3 and HUES7 at similar passage. We generated inferred interactome networks using transcriptomic data unique to the ICM or TE, and defined a hierarchy of modules (highly connected regions with shared function). We compared network properties and the modular hierarchy and show that the three hESCs had limited overlap with ICM specific transcriptome (6%-12%). However this overlap was enriched for network properties related to transcriptional activity in ICM (p=0.016); greatest in MAN1 compared to HUES3 (p=0.048) or HUES7 (p=0.012). The hierarchy of modules in the ICM interactome contained a greater proportion of MAN1 specific gene expression (46%) compared to HUES3 (28%) and HUES7 (25%) (p=9.0×10−4).These findings show that traditional methods based on transcriptome overlap are not sufficient to identify divergence of hESCs from ICM. Our approach also provides a valuable approach to the quantification of differences between hESC lines.And Manchester Academic Health Sciences Centre

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