scholarly journals Live Fluorescent RNA-Based Detection of Pluripotency Gene Expression in Embryonic and Induced Pluripotent Stem Cells of Different Species

Stem Cells ◽  
2015 ◽  
Vol 33 (2) ◽  
pp. 392-402 ◽  
Author(s):  
Harald Lahm ◽  
Stefanie Doppler ◽  
Martina Dreßen ◽  
Astrid Werner ◽  
Klaudia Adamczyk ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Pluripotency Gene ◽  
Induced Pluripotent

scholarly journals T9. EPIGENETIC PROFILING IN SCHIZOPHRENIA DERIVED HUMAN INDUCED PLURIPOTENT STEM CELLS (HIPSCS) AND NEURONS

2020 ◽  
Vol 46 (Supplement_1) ◽  
pp. S234-S234
Author(s):  
Lorna Farrelly ◽  
Shuangping Zhang ◽  
Erin Flaherty ◽  
Aaron Topol ◽  
Nadine Schrode ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mass Spectrometry ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Neural Progenitor Cells ◽  
Gene Expression Pattern ◽  
Early Gene ◽  
Label Free ◽  
Induced Pluripotent

Abstract Background Schizophrenia (SCZ) is a severe psychiatric disorder affecting ~1% of the world’s population. It is largely heritable with genetic risk reflected by a combination of common variants of small effect and highly penetrant rare mutations. Chromatin modifications are known to play critical roles in the mediation of many neurodevelopmental processes, and, when disturbed, may also contribute to the precipitation of psychiatric disorders, such as SCZ. While a handful of candidate-based studies have measured changes in promoter-bound histone modifications, few mechanistic studies have been carried out to explore how these modifications may affect chromatin to precipitate behavioral phenotypes associated with the disease. Methods We applied an unbiased proteomics approach to evaluate the epigenetic landscape of SCZ in human induced pluripotent stem cells (hiPSC), neural progenitor cells (NPCs) and neurons from SCZ patients vs. matched controls. We utilized proteomics-based, label free liquid chromatography mass spectrometry (LC-MS/MS) on purified histones from these cells and confirmed our results by western blotting in postmortem SCZ cortical brain tissues. Furthermore we validated our findings with the application of histone interaction assays and structural and biophysical assessments to identify and confirm novel chromatin ‘readers’. To relate our findings to a SCZ phenotype we used a SCZ rodent model of prepulse inhibition (PPI) to perform pharmacological manipulations and behavioral assessments. Results Using label free mass spectrometry we performed PTM screening of hiPSCs, NPCs and matured neurons derived from SCZ patients and matched controls. We identified, amongst others, altered patterns of hyperacetylation in SCZ neurons. Additionally we identified enhanced binding of particular acetylation ‘reader’ proteins. Pharmacological inhibition of such proteins in an animal model of amphetamine sensitization ameliorated PPI deficits further validating this epigenetic signature in SCZ. Discussion Recent evidence indicates that relevance and patterns of acetylation in epigenetics advances beyond its role in transcription and small molecule inhibitors of these aberrant interactions hold promise as useful therapeutics. This study identifies a role for modulating gene expression changes associated with a SCZ epigenetic signature and warrants further investigation in terms of how this early gene expression pattern perhaps determines susceptibility or severity of the SCZ disease trajectory.

scholarly journals Preferential gene expression and epigenetic memory of induced pluripotent stem cells derived from mouse pancreas

2015 ◽  
Vol 20 (5) ◽  
pp. 367-381 ◽  
Author(s):  
Daiki Nukaya ◽  
Kohtaro Minami ◽  
Ritsuko Hoshikawa ◽  
Norihide Yokoi ◽  
Susumu Seino
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Epigenetic Memory ◽  
Mouse Pancreas ◽  
Induced Pluripotent

15q11.2 deletion is enriched in patients with total anomalous pulmonary venous connection

2020 ◽  
pp. jmedgenet-2019-106608
Author(s):  
Xiaoliang Li ◽  
Guocheng Shi ◽  
Yang Li ◽  
Xiaoqing Zhang ◽  
Ying Xiang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Mononuclear Cells ◽  
Chromosomal Microarray ◽  
Cardiomyocyte Differentiation ◽  
Induced Pluripotent ◽  
Anomalous Pulmonary Venous Connection ◽  
15Q11.2 Deletion

IntroductionCNV is a vital pathogenic factor of congenital heart disease (CHD). However, few CNVs have been reported for total anomalous pulmonary venous connection (TAPVC), which is a rare form of CHD. Using case-control study, we identified 15q11.2 deletion associated with TAPVC. We then used a TAPVC trio as model to reveal possible molecular basis of 15q11.2 microdeletion.MethodsCNVplex and Chromosomal Microarray were used to identify and validate CNVs in samples from 231 TAPVC cases and 200 healthy controls from Shanghai Children’s Medical Center. In vitro cardiomyocyte differentiation of induced pluripotent stem cells from peripheral blood mononuclear cells for a TAPVC trio with paternal inherited 15q11.2 deletion was performed to characterise the effect of the deletion on cardiomyocyte differentiation and gene expression.ResultsThe 15q11.2 microdeletion was significantly enriched in patients with TAPVC compared with healthy control (13/231 in patients vs 0/200 in controls, p=5.872×10−2, Bonferroni adjusted) using Fisher’s exact test. Induced pluripotent stem cells from the proband could not differentiate into normal cardiomyocyte. Transcriptomic analysis identified a number of differentially expressed genes in the 15q11.2 deletion carriers of the family. TAPVC disease-causing genes such as PITX2, NKX2-5 and ANKRD1 showed significantly higher expression in the proband compared with her healthy mother. Knockdown of TUBGCP5 could lead to abnormal cardiomyocyte differentiation.ConclusionWe discovered that the 15q11.2 deletion is significantly associated with TAPVC. Gene expression profile that might arise from 15q11.2 deletion for a TAPVC family was characterised using cell experiments.

scholarly journals Modelling the developmental spliceosomal craniofacial disorder Burn-McKeown syndrome using induced pluripotent stem cells

2020 ◽  
Author(s):  
Katherine A. Wood ◽  
Charlie F. Rowlands ◽  
Huw B. Thomas ◽  
Steven Woods ◽  
Julieta O’Flaherty ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Craniofacial Development ◽  
Rna Seq ◽  
Unrelated Control ◽  
Mesenchymal Transition ◽  
Base Pair Deletion ◽  
Induced Pluripotent

ABSTRACTThe craniofacial developmental disorder Burn-McKeown Syndrome (BMKS) is caused by biallelic variants in the pre-messenger RNA splicing factor gene TXNL4A/DIB1. The majority of affected individuals with BMKS have a 34 base pair deletion in the promoter region of one allele of TXNL4A combined with a loss-of-function variant on the other allele, resulting in reduced TXNL4A expression. However, it is unclear how reduced expression of this ubiquitously expressed spliceosome protein results in craniofacial defects during development. Here we reprogrammed peripheral mononuclear blood cells from a BMKS patient and her unaffected mother into induced pluripotent stem cells (iPSCs) and differentiated the iPSCs into induced neural crest cells (iNCCs), the key cell type required for correct craniofacial development. BMKS patient-derived iPSCs proliferated more slowly than both mother- and unrelated control-derived iPSCs, and RNA-Seq analysis revealed significant differences in gene expression and alternative splicing. Patient iPSCs displayed defective differentiation into iNCCs compared to maternal and unrelated control iPSCs, in particular a delay in undergoing an epithelial-to-mesenchymal transition (EMT). RNA-Seq analysis of differentiated iNCCs revealed widespread gene expression changes and mis-splicing in genes relevant to craniofacial and embryonic development that highlight a dampened response to WNT signalling, the key pathway activated during iNCC differentiation. Furthermore, we identified the mis-splicing of TCF7L2 exon 4, a key gene in the WNT pathway, as a potential cause of the downregulated WNT response in patient cells. Additionally, mis-spliced genes shared common sequence properties such as length, splice site strengths and sequence motifs, suggesting that splicing of particular subsets of genes is particularly sensitive to changes in TXNL4A expression. Together, these data provide the first insight into how reduced TXNL4A expression in BMKS patients might compromise splicing and NCC function, resulting in defective craniofacial development in the embryo.

scholarly journals Comparative Analysis of Tenogenic Gene Expression in Tenocyte-Derived Induced Pluripotent Stem Cells and Bone Marrow-Derived Mesenchymal Stem Cells in Response to Biochemical and Biomechanical Stimuli

2021 ◽  
Vol 2021 ◽  
pp. 1-18
Author(s):  
Feikun Yang ◽  
Dean W. Richardson
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ectopic Expression ◽  
Mechanical Stretch ◽  
Specific Gene ◽  
Induced Pluripotent

The tendon is highly prone to injury, overuse, or age-related degeneration in both humans and horses. Natural healing of injured tendon is poor, and cell-based therapeutic treatment is still a significant clinical challenge. In this study, we extensively investigated the expression of tenogenic genes in equine bone marrow mesenchymal stem cells (BMSCs) and tenocyte-derived induced pluripotent stem cells (teno-iPSCs) stimulated by growth factors (TGF-β3 and BMP12) combined with ectopic expression of tenogenic transcription factor MKX or cyclic uniaxial mechanical stretch. Western blotting revealed that TGF-β3 and BMP12 increased the expression of transcription factors SCX and MKX in both cells, but the tenocyte marker tenomodulin (TNMD) was detected only in BMSCs and upregulated by either inducer. On the other hand, quantitative real-time PCR showed that TGF-β3 increased the expression of EGR1, COL1A2, FMOD, and TNC in BMSCs and SCX, COL1A2, DCN, FMOD, and TNC in teno-iPSCs. BMP12 treatment elevated SCX, MKX, DCN, FMOD, and TNC in teno-iPSCs. Overexpression of MKX increased SCX, DCN, FMOD, and TNC in BMSCs and EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-β3 further enhanced TNC in BMSCs. Moreover, mechanical stretch increased SCX, EGR1, DCN, ELN, and TNC in BMSCs and SCX, MKX, EGR1, COL1A2, DCN, FMOD, and TNC in teno-iPSCs; TGF-β3 tended to further elevate SCX, ELN, and TNC in BMSCs and SCX, MKX, COL1A2, DCN, and TNC in teno-iPSCs, while BMP12 further uptrended the expression of SCX and DCN in BMSCs and DCN in teno-iPSCs. Additionally, the aforementioned tenogenic inducers also affected the expression of signaling regulators SMAD7, ETV4, and SIRT1 in BMSCs and teno-iPSCs. Taken together, our data demonstrate that, in respect to the tenocyte-lineage-specific gene expression, BMSCs and teno-iPSCs respond differently to the tenogenic stimuli, which may affect the outcome of their application in tendon repair or regeneration.

scholarly journals Evaluation of ATG5 Gene Expression in Human Induced Pluripotent Stem Cells During Endoderm Induction

2020 ◽  
Vol In Press (In Press) ◽  
Author(s):  
Alice Sabet ◽  
Negar Azarpira ◽  
Saeid Ghavami ◽  
Leila Kohan
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Mrna Expression ◽  
Pluripotent Stem Cells ◽  
Embryoid Body ◽  
Metabolic Stress ◽  
High Rate ◽  
Human Ipscs ◽  
Induced Pluripotent

Background: Autophagy is a vital cell survival mechanism that authorizes cells to assort to metabolic stress and is essential for the development and maintenance of cellular and tissue homeostasis, as well as the prevention of human disease. It has also been shown that autophagy plays a significant role in the development and differentiation of stem cells, as well as induced pluripotent stem cells (iPSCs). Objectives: The present study aimed to examine the mRNA expression of the ATG5 gene, one of the key markers of autophagy in human iPSCs (hiPSCs) during endoderm induction. Methods: In this study, we cultured the human iPSC line (R1-hiPSC1) on mitomycin-C, inactivated mouse embryonic fibroblasts (MEF) layer, and used hanging drop protocol to generate embryoid body (EB) and expose differentiation. The Real-time PCR method was used to examine the mRNA expression level of ATG5 in hiPSC during endoderm induction. Results: Our results demonstrated the high mRNA expression of ATG5 in the MEI stage, which shows the high rate of autophagy in MEI days rather than the other stages of differentiation. Conclusions: The modification of ATG5 gene expression within hiPSC during endoderm induction shows the importance of autophagy assessments in hiPSC differentiation. Therefore, subsequent studies are needed to clarify the details of autophagy effects on hiPSC differentiation.

223 ISOLATION OF RABBIT PLURIPOTENCY GENES TO GENERATE RABBIT INDUCED PLURIPOTENT STEM CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 223 ◽  
Author(s):  
Z. Tancos ◽  
O. Ujhelly ◽  
M. K. Pirity ◽  
A. Dinnyes
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Somatic Cells ◽  
Blastocyst Stage ◽  
Promising Tool ◽  
Human Genes ◽  
Pluripotency Gene ◽  
Induced Pluripotent ◽  
Pluripotency Genes

Induced pluripotent stem cells (iPSC) technology, which allows direct reprogramming of somatic cells to a pluripotent state, is a promising tool for gene-function studies disease modelling, drug screening, toxicology tests and to generate knockout animal models. The goal of the current work was to close the gap in knowledge with regard to the molecular biological background for rabbit iPS work by isolating the putative pluripotency genes from the rabbit, based on the sequences published for other species. The sequence of known pluripotency genes (Oct4, Sox2, Klf4, c-Myc, Nanog) were analysed and primers designed based on the similarity of sequences. Sequences of each individual rabbit pluripotency gene was compared to other vertebrates (e.g. human, mouse, bovine) phylogenetically. Rabbit ESCs and blastocyst stage embryos were collected from superovulated rabbits to isolate total RNA. Genes of interest were amplified using RT-PCR and electrophoretically separated for cDNA fragment isolation. Isolated and subcloned cDNA fragments were sequenced and analysed. Our results showed that after restriction digestion the size of amplified and cloned rabbit Oct4, Sox2, Klf4, c-Myc and Nanog gene fragments correspond to the expected amplicon size. Furthermore, sequence confirmation by DNA sequencing has been completed in the case of Oct4, c-Myc, Klf4 and Nanog. The homology of these genes to that of their mouse and human orthologs were as follows: Oct4: at Na level 79% homologue to mouse, 85% homologue to human, at Aa level 81% homologue to mouse, 90% homologue to human; Klf4: at Na level 98% homologue to mouse, 85% homologue to human, at Aa level 95% homologue to mouse, 84% homologue to human; c-myc: at Na level 88% homologue to mouse, 92% homologue to human, at Aa level 91% homologue to mouse and 94% homologue to human; Nanog: at Na level 71% homologue to mouse, 78% homologue to human, at Aa level 55% homologue to mouse, 66% homologue to human. In conclusion, we have revealed differences at both Na and Aa level in all four major rabbit pluripotency gene sequences in comparison to their mammalian orthologs which might partially explain difficulties in generation of rabbit iPSC capable of germline transmission. Our further goal is to apply rabbit specific pluripotency genes to reprogram somatic cells and generate iPSC more efficiently than by using mouse or human genes. This work was supported by grants from Plurabbit, OMFB-00130/2010 ANR-NKTH; NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; EU FP7 (AniStem, PIAP-GA-2011-286264PartnErS, PIAP-GA-2008-218205; InduStem, PIAP-GA-2008-230675; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; PluriSys, HEALTH-2007-B-223485).

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