scholarly journals Generation and validation of a PITX2–EGFP reporter line of human induced pluripotent stem cells enables isolation of periocular mesenchymal cells

2020 ◽  
Vol 295 (11) ◽  
pp. 3456-3465 ◽  
Author(s):  
Toru Okubo ◽  
Ryuhei Hayashi ◽  
Shun Shibata ◽  
Yuji Kudo ◽  
Yuki Ishikawa ◽  
...  
Keyword(s):  
Stop Codon ◽  
Marker Gene ◽  
Induced Pluripotent Stem Cell ◽  
Mesenchymal Cells ◽  
Immunofluorescence Staining ◽  
Mrna Levels ◽  
Transcription Activator ◽  
Fluorescent Reporter ◽  
Induced Pluripotent ◽  
Pitx2 Expression

PITX2 (Paired-like homeodomain transcription factor 2) plays important roles in asymmetric development of the internal organs and symmetric development of eye tissues. During eye development, cranial neural crest cells migrate from the neural tube and form the periocular mesenchyme (POM). POM cells differentiate into several ocular cell types, such as corneal endothelial cells, keratocytes, and some ocular mesenchymal cells. In this study, we used transcription activator–like effector nuclease technology to establish a human induced pluripotent stem cell (hiPSC) line expressing a fluorescent reporter gene from the PITX2 promoter. Using homologous recombination, we heterozygously inserted a PITX2–IRES2–EGFP sequence downstream of the stop codon in exon 8 of PITX2. Cellular pluripotency was monitored with alkaline phosphatase and immunofluorescence staining of pluripotency markers, and the hiPSC line formed normal self-formed ectodermal autonomous multizones. Using a combination of previously reported methods, we induced PITX2 in the hiPSC line and observed simultaneous EGFP and PITX2 expression, as indicated by immunoblotting and immunofluorescence staining. PITX2 mRNA levels were increased in EGFP-positive cells, which were collected by cell sorting, and marker gene expression analysis of EGFP-positive cells induced in self-formed ectodermal autonomous multizones revealed that they were genuine POM cells. Moreover, after 2 days of culture, EGFP-positive cells expressed the PITX2 protein, which co-localized with forkhead box C1 (FOXC1) protein in the nucleus. We anticipate that the PITX2–EGFP hiPSC reporter cell line established and validated here can be utilized to isolate POM cells and to analyze PITX2 expression during POM cell induction.

The Influence of FLI1 Expression Levels on Megakaryopoiesis: Studies Using iPSCs

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2352-2352
Author(s):  
Karen K Vo ◽  
Danuta Jadwiga Jarocha ◽  
Randolph B Lyde ◽  
Spencer Sullivan ◽  
Deborah French ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Induced Pluripotent Stem Cell ◽  
Allelic Exclusion ◽  
Control Line ◽  
Mrna Levels ◽  
Specific Expression ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Induced Pluripotent ◽  
And Control

Abstract Jacobsen syndrome is a rare, inherited hemizygous deletion of chromosome 11q that is often associated with a dysmegakaryopoiesis and macrothrombocytopenia termed Paris Trousseau syndrome (PTSx). Among the genes involved in the chromosomal deletion are FLI1 and ETS1, both of which belong to the ETS family of transcription factors and have been associated with megakaryopoiesis. One prior study using primary human hematopoietic stem cells suggested that the defect in PTSx was due to FLI1 allelic exclusion resulting in the generation of two distinct megakaryocyte (MK) populations, one immature and one mature. More consistent with the clinical course of this disorder, we hypothesize that PTSx is caused by FLI1 haploinsufficiency, where all MKs are affected and do not mature properly. The goal of our studies was to better understand MK development by investigating the role of FLI1 during megakaryopoiesis, including in PTSx. However, Fli1 deletions in mice did not replicate the defect observed in humans, so we used genome-engineered human induced pluripotent stem cell (iPSC) lines. We have established an iPSC line from a PTSx patient and derived from this a FLI1 overexpressing (OE) line in which FLI1 cDNA was cloned into the AAVS1 "safe harbor" locus with MK-specific expression driven by the GP1bα promoter. In parallel, we have a healthy control line, a control-derived FLI1 OE line, and a homozygous FLI1+/- line. In the control- and PTSx-FLI1 OE lines, FLI1 mRNA levels in MKs were 2X higher than control levels. The FLI1+/- line was generated using TALENs and expressed RNA at levels comparable to the PTSx line. To analyze MK progenitor potential, the iPSC lines were differentiated to hematopoietic progenitor cells (HPCs) and analyzed using Megacult colony assays. The PTSx line generated 4- to 6-fold less CFU-MK colonies per 1000 plated CD41+CD235+ cells compared to control (P=0.1) and PTSx-FLI1 OE (P=0.002). Likewise, the FLI1+/- line had less colonies compared to control (P=0.2) and control-FLI1 OE (P=0.005). The control-FLI1 OE line generated 70% more colonies (P=0.22) than the control line. To analyze MK generation, identical numbers of HPCs were expanded in liquid culture containing MK-specific cytokines and the numbers of CD41+CD42a+ cells were quantitated. The PTSx line had <20% the number of MKs generated in the control. The numbers of MKs generated from the PTSx-FLI1 OE and FLI1+/- HPCs were about half that of control line, while the number of MKs generated from the control FLI1 OE line was 40% higher than control. Platelet function studies show that CD42b+ PTSx and FLI1+/- platelet-like particles (PLPs) were unresponsive to convulxin stimulation compared to CD42b+ control, control-FLI1 OE, and PTSx-FLI1 OE PLPs. In addition, the PTSx and FLI1+/- MKs began to lose CD42b after only 3 days in culture while the PTSx-FLI1 OE and control MKs began to lose CD42b after 6 days in culture. The control-FLI1 OE MKs still retained CD42b expression after 8 days in culture. Overall, these data support our hypothesis that FLI1 haploinsufficiency underlies PTSx, as two distinct MK populations were not observed. Furthermore, the FLI1+/- MKs had similar characteristics to the PTSx-derived MKs, which pinpoints FLI1 deletion as the cause of PTSx MK deficiency. More importantly, we show that MK commitment of HPCs, MK expansion and maturation, their ability to retain CD42b expression and response to agonist stimulation correlate with FLI1 expression levels. Our findings have implications for production of functional MKs and platelets for future clinical application. Disclosures No relevant conflicts of interest to declare.

scholarly journals Control of Microbial Opsin Expression in Stem Cell Derived Cones for Improved Outcomes in Cell Therapy

2021 ◽  
Vol 15 ◽  
Author(s):  
Marcela Garita-Hernandez ◽  
Antoine Chaffiol ◽  
Laure Guibbal ◽  
Fiona Routet ◽  
Hanen Khabou ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cell Therapy ◽  
Transgene Expression ◽  
Induced Pluripotent Stem Cell ◽  
Fluorescent Reporter ◽  
Human Organ ◽  
Gene Therapies ◽  
Transcriptional Changes ◽  
A Cell ◽  
Induced Pluripotent

Human-induced pluripotent stem cell (hiPSC) derived organoids have become increasingly used systems allowing 3D-modeling of human organ development, and disease. They are also a reliable source of cells for transplantation in cell therapy and an excellent model to validate gene therapies. To make full use of these systems, a toolkit of genetic modification techniques is necessary to control their activity in line with the downstream application. We have previously described adeno-associated viruse (AAV) vectors for efficient targeting of cells within human retinal organoids. Here, we describe biological restriction and enhanced gene expression in cone cells of such organoids thanks to the use of a 1.7-kb L-opsin promoter. We illustrate the usefulness of implementing such a promoter to enhance the expression of the red-shifted opsin Jaws in fusion with a fluorescent reporter gene, enabling cell sorting to enrich the desired cell population. Increased Jaws expression after transplantation improved light responses promising better therapeutic outcomes in a cell therapy setting. Our results point to the importance of promoter activity in restricting, improving, and controlling the kinetics of transgene expression during the maturation of hiPSC retinal derivatives. Differentiation requires mechanisms to initiate specific transcriptional changes and to reinforce those changes when mature cell states are reached. By employing a cell-type-specific promoter we put transgene expression under the new transcriptional program of mature cells.

scholarly journals Transcriptional profiling of isogenic Friedreich ataxia induced pluripotent stem cell-derived neurons

2018 ◽  
Author(s):  
Jiun-I Lai ◽  
Daniel Nachun ◽  
Lina Petrosyan ◽  
Benjamin Throesch ◽  
Erica Campau ◽  
...  
Keyword(s):  
Nervous System ◽  
Stem Cell ◽  
Sensory Neurons ◽  
Pluripotent Stem Cell ◽  
Neuronal Cells ◽  
Friedreich Ataxia ◽  
Induced Pluripotent Stem Cell ◽  
Repeat Expansion ◽  
Mrna Levels ◽  
Induced Pluripotent

AbstractFriedreich ataxia (FRDA) is a rare childhood neurodegenerative disorder with no effective treatment. FRDA is caused by transcriptional silencing of the FXN gene and consequent loss of the essential mitochondrial protein frataxin. Based on the knowledge that a GAA•TTC repeat expansion in the first intron of FXN leads to heterochromatin formation and gene silencing, we have shown that members of the 2-aminobenzamide family of histone deacetylase inhibitors (HDACi) reproducibly increase FXN mRNA levels in induced pluripotent stem cell (iPSC)-derived FRDA neuronal cells and in peripheral blood mononuclear cells from patients treated with the drug in a phase I clinical trial. How the reduced expression of frataxin leads to neurological and other systemic symptoms in FRDA patients remains unclear. Similarly to other triplet repeat disorders, it is not known why only specific cells types are affected in the disease, primarily the large sensory neurons of the dorsal root ganglia and cardiomyocytes. The combination of iPSC technology and genome editing techniques offers the unique possibility of addressing these questions in a relevant cell model of the disease, without the confounding effect of different genetic backgrounds. We derived a set of isogenic iPSC lines that differ only in the length of the GAA•TTC repeats, using “scarless” gene-editing methods (helper-dependent adenovirus-mediated homologous recombination). To uncover the gene expression signature due to GAA•TTC repeat expansion in FRDA neuronal cells and the effect of HDACi on these changes, we performed transcriptomic analysis of iPSC-derived central nervous system (CNS) and isogenic sensory neurons by RNA sequencing. We find that multiple cellular pathways are commonly affected by the loss of frataxin in CNS and peripheral nervous system neurons and these changes are partially restored by HDACi treatment.

scholarly journals Immunomodulatory Properties of Induced Pluripotent Stem Cell-Derived Mesenchymal Cells

2016 ◽  
Vol 117 (12) ◽  
pp. 2844-2853 ◽  
Author(s):  
Jia Ng ◽  
Kim Hynes ◽  
Gregory White ◽  
Kisha Nandini Sivanathan ◽  
Kate Vandyke ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Mesenchymal Cells ◽  
Immunomodulatory Properties ◽  
Induced Pluripotent

scholarly journals Inhibition of ceramide synthesis improves the outcome in ischemia/reperfusion injury using human-induced pluripotent stem cell derived cardiomyocyte

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
P Haxhikadrija ◽  
M Bekhite ◽  
T Kretzschmar ◽  
J Wu ◽  
A Maloku ◽  
...  
Keyword(s):  
Stem Cell ◽  
Reperfusion Injury ◽  
Pluripotent Stem Cell ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Induced Pluripotent Stem Cell ◽  
Calcium Oscillations ◽  
Mrna Levels ◽  
Ceramide Synthesis ◽  
Induced Pluripotent

Abstract Introduction and aim Ceramides are proven to be biologically active in apoptosis, inflammation, mitochondrial dysfunction, and as a second messenger in various signaling pathways1. However, the data linking the role of ceramides in ischemia/reperfusion injury (I/R injury) are lacking. We aimed to establish an I/R injury model using human-induced pluripotent stem cell (hiPSC)-derived cardiomyocyte (CM) and to evaluate ceramide levels, ceramide synthesis pathway, and outcome of CM with inhibition of ceramide synthesis during I/R injury. Methods HiPSC technology has been used to generate functional human CMs to elucidate the underlying mechanisms of the pathophysiology of the human heart. Results In our model, we observed an increase of mRNA levels of genes regulating ceramide synthesis after 6 h of ischemia followed by 16 h reperfusion, such as SPTLC1 (1.1±0.08 vs 1.0, p=0.2), CerS2 (1.6±0.3 vs 1.0, p&lt;0.001), CerS4 (1.3±0.1 vs 1.0, p=0.02), CerS5 (1.3±0.1 vs 1.0, p=0.03), and SMPD (1.6±0.1 vs 1.0, p=0.008) compared to control. Also, both long- and very long-chain ceramide species levels measured with mass spectrometry were increased significantly after 6 h ischemia followed by 16 h reperfusion compared to control (C14:0: 1,1±0.3 pmol/million cells vs 0,3±0,2 pmol/ million cells, p=0.02 and C24:1: 26,3±7,1 pmol/ million cells vs 9,6±3,4 pmol/ million cells, p=0.02). Inhibition of ceramide synthesis with Fumonisin B1 (FB1) significantly increased the viability after 6h of ischemia followed by 16 h of reperfusion compared to CMs incubated without inhibitors (32.2%±1.5% vs 26.9%±2.6%, p=0.04). Interestingly, we identified two mechanisms with which the viability improves after incubation with ceramide inhibitor. The first mechanism observed could be the restoration of both intracellular calcium baseline (control 29±1.2, I/R 55±5.7 and I/R with FB1 35.6±2.5, p&lt;0,001) and peak (control 45.1±5.6, I/R 94.3±5.7 and I/R with FB1 56.5±7.5, p&lt;0,001) levels to nearly the same levels as observed in control samples. A possible cause of increased calcium oscillations after 6 h of ischemia followed by 3 h of reperfusion in the first place could be an upregulation of the RyR2 levels detected by qPCR (2.5±0.4 vs control 1.0, p=0.008). The second mechanism of improving viability in I/R injury could be a decrease of generation of reactive oxygen species (ROS) detected by MitoSOX dye after incubation with FB1 inhibitor to nearly the same levels as observed in control (control 22±5.1, I/R 33.8±5.8 and I/R with FB1 30.7±5.9, p=0,06). Conclusion We conclude that ceramides have important implications in either mediating or causing injury and their inhibition improves the outcome of I/R injury by decreasing ROS generation and improving calcium oscillations. FUNDunding Acknowledgement Type of funding sources: Public hospital(s). Main funding source(s): Jena University Hospital, Clinic for Internal Medicine 1Interdisciplinary Center for Clinical Research Jena

scholarly journals Protein and mRNA Quantification in Small Samples of Human Induced Pluripotent Stem-cell-derived Cardiomyocytes in 96-well Microplates

2021 ◽  
Author(s):  
Weizhen Li ◽  
Julie Han ◽  
Emilia Entcheva
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Protein Detection ◽  
Small Sample ◽  
Protein Quantification ◽  
Small Samples ◽  
Mrna Levels ◽  
Mrna Quantification ◽  
Induced Pluripotent

We describe a method for protein quantification and for mRNA quantification in small sample quantities of human induced pluripotent stem-cell-derived-cardiomyocytes (hiPSC-CMs). Demonstrated here is how the capillary-based protein detection system WesTM by ProteinSimple and the Power SYBRTM Green Cells-to-CTTM Kit by Invitrogen can be applied to individual samples in a 96-well microplate format and thus made compatible with high-throughput (HT) cardiomyocyte assays. As an example of the usage, we illustrate that Cx43 protein and GJA1 mRNA levels in hiPSC-CMs are enhanced when the optogenetic actuator, channelrodopsin-2 (ChR2), is genetically expressed in them. Instructions are presented for cell culture and lysate preparations from hiPSC-CMs, along with optimized parameter settings and experimental protocol steps. Strategies to optimize primary antibody concentrations as well as ways for signal normalization are discussed, i.e. antibody multiplexing and total protein assay. The sensitivity of both the Wes and Cells-to-CT kit enables protein and mRNA quantification in a HT format, which is important when dealing with precious small samples. In addition to being able to handle small cardiomyocyte samples, these streamlined and semi-automated processes enable quick mechanistic analysis.

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