scholarly journals High-level transgene expression in induced pluripotent stem cell–derived megakaryocytes: correction of Glanzmann thrombasthenia

Blood ◽  
2014 ◽  
Vol 123 (5) ◽  
pp. 753-757 ◽  
Author(s):  
Spencer K. Sullivan ◽  
Jason A. Mills ◽  
Sevasti B. Koukouritaki ◽  
Karen K. Vo ◽  
Randolph B. Lyde ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Type I ◽  
Glanzmann Thrombasthenia ◽  
Key Points ◽  
Single Allele ◽  
Induced Pluripotent ◽  
Aavs1 Locus ◽  
High Level

Key Points When targeted to a single allele of the AAVS1 locus, the Gp1ba promoter drives a high level of expression specifically to megakaryocytes. Transgene rescue in iPSCs provides a model for the return of surface αIIbβ3 expression to near-normal levels in patients with type I GT.

Tissue-Specific Transgene Expression in Induced Pluripotent Stem (iPS) Cell-Derived Megakaryocytes: Correction of Glanzmann Thrombasthenia (GT)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 387-387
Author(s):  
Spencer K. Sullivan ◽  
Jason A. Mills ◽  
Li Zhai ◽  
Prasuna Paluru ◽  
Karen Vo ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Surface Expression ◽  
Ips Cells ◽  
Severe Bleeding ◽  
Mrna Levels ◽  
Ips Cell ◽  
Platelet Disorder ◽  
Single Allele ◽  
Aavs1 Locus ◽  
High Level

Abstract Abstract 387 Megakaryocyte-specific transgene expression in patient-derived iPS cells offers a new approach to study and potentially treat disorders that affect megakaryocytes and platelets. The feasibility of this method was demonstrated in GT, a disorder resulting from an absence of functional platelet integrin alphaIIb/beta3, leading to impaired platelet aggregation and clinically presenting with severe bleeding. While rare, GT can serve as a model of an inherited platelet disorder that would benefit from corrective cell therapy. For this study, iPS cells were generated from the peripheral blood of healthy controls and two patients with GT. Patient 1 is homozygous for a novel alphaIIb exon/intron 4 splice site mutation, leading to an alternatively spliced transcript and premature stop codon. Patient 2 is homozygous for a previously reported alphaIIb missense mutation in exon 8 (p.G273D), leading to impaired intracellular transport and surface expression of the heterodimer. Both patients express <5% alphaIIb/beta3 on the surface of their platelets, which was confirmed in IPS cell-derived megakaryocytes. To achieve megakaryocyte-specific expression, transgene constructs were created using the murine proximal GPIbalpha promoter driving either an eGFP reporter or wild type alphaIIb cDNA. These constructs were inserted into the AAVS1 safe harbor locus using zinc finger nuclease-mediated homologous recombination. When targeted to a single allele of the AAVS1 locus in human ES cells, the GPIbalpha promoter driving eGFP showed a high-level of expression in ES cell-derived megakaryocytes. Inserting the GPIbalpha promoter driving alphaIIb into a single allele of the AAVS1 locus in GT iPS cells restored ∼60% surface expression of integrin alphaIIb/beta3 when compared to control iPS cell-derived megakaryocytes. Transgenic alphaIIb mRNA levels were similar to endogenous alphaIIb mRNA levels in differentiated megakaryocytes. When stimulated with thrombin, corrected GT iPS cell-derived megakaryocytes demonstrated both PAC-1 antibody binding and fibrinogen binding. This study shows the GPIbalpha promoter construct targeted to the AAVS1 locus drives a high-level of expression in iPS cell-derived megakaryocytes, which offers a novel tool to study megakaryocyte and platelet biology in samples obtained from affected individuals. For GT, this study demonstrates a higher level of correction than seen with a lentiviral approach. To our knowledge, this is the first report of the generation and correction of iPS cell lines from patients with a disease affecting platelet function. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Characterization of Functional Human Skeletal Myotubes and Neuromuscular Junction Derived—From the Same Induced Pluripotent Stem Cell Source

2020 ◽  
Vol 7 (4) ◽  
pp. 133
Author(s):  
Xiufang Guo ◽  
Agnes Badu-Mensah ◽  
Michael C. Thomas ◽  
Christopher W. McAleer ◽  
James J. Hickman
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
Neuromuscular Junctions ◽  
Functional Characterization ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Type I ◽  
Induced Pluripotent

In vitro generation of functional neuromuscular junctions (NMJs) utilizing the same induced pluripotent stem cell (iPSC) source for muscle and motoneurons would be of great value for disease modeling and tissue engineering. Although, differentiation and characterization of iPSC-derived motoneurons are well established, and iPSC-derived skeletal muscle (iPSC-SKM) has been reported, there is a general lack of systemic and functional characterization of the iPSC-SKM. This study performed a systematic characterization of iPSC-SKM differentiated using a serum-free, small molecule-directed protocol. Morphologically, the iPSC-SKM demonstrated the expression and appropriate distribution of acetylcholine, ryanodine and dihydropyridine receptors. Fiber type analysis revealed a mixture of human fast (Type IIX, IIA) and slow (Type I) muscle types and the absence of animal Type IIB fibers. Functionally, the iPSC-SKMs contracted synchronously upon electrical stimulation, with the contraction force comparable to myofibers derived from primary myoblasts. Most importantly, when co-cultured with human iPSC-derived motoneurons from the same iPSC source, the myofibers contracted in response to motoneuron stimulation indicating the formation of functional NMJs. By demonstrating comparable structural and functional capacity to primary myoblast-derived myofibers, this defined, iPSC-SKM system, as well as the personal NMJ system, has applications for patient-specific drug testing and investigation of muscle physiology and disease.

scholarly journals Induced pluripotent stem cell–based mapping of β-globin expression throughout human erythropoietic development

2018 ◽  
Vol 2 (15) ◽  
pp. 1998-2011 ◽  
Author(s):  
Kim Vanuytsel ◽  
Taylor Matte ◽  
Amy Leung ◽  
Zaw Htut Naing ◽  
Tasha Morrison ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Expression Profile ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Yolk Sac ◽  
Biological Processes ◽  
Erythroid Cells ◽  
Key Points ◽  
Induced Pluripotent

Key Points iPSC-derived definitive erythroid cells display a globin expression profile corresponding to yolk sac erythromyeloid progenitors. iPSC-derived erythroblasts resemble their postnatal counterparts in terms of gene expression and essential biological processes.

scholarly journals Angiotensin II induces apoptosis in human induced pluripotent stem cell-derived cardiomyocytes

2020 ◽  
Author(s):  
Qiang Gao ◽  
Ping Wang ◽  
Zhiming Wu ◽  
Hailong Qiu ◽  
Bin Lin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Angiotensin Ii ◽  
Pluripotent Stem Cell ◽  
Heart Diseases ◽  
Day Treatment ◽  
Induced Pluripotent Stem Cell ◽  
Type I ◽  
High Concentration ◽  
Long Term Treatment ◽  
Induced Pluripotent

AbstractBackgroundThe renin-angiotensin system (RAS) functions fundamentally to regulate the pathological process of cardiovascular diseases, such as heart failure and hypertension. As the major effector in RAS, angiotensin II activates angiotensin II receptors to initiate the downstream pathways, which lead to the phenotypes including apoptosis, hypertrophy, and cardiac remodeling. Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) are being applied as a promising platform for personalized medicine to heart diseases. However, whether angiotensin II induces apoptosis in iPSC-CM is still obscure, which raises an uncertainty about the clinical applications of iPSC-CM.MethodsWe treated iPSC-CM with angiotensin II at eight concentrations (0 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM and 1 mM) and four incubation durations (24 hours, 48 hours, 6 days and 10 days), then PrestoBlue reagent and a apoptosis marker were used to examine the viability and apoptosis status of cardiomyocytes from each group. The expression levels of some apoptosis and proliferation related genes were also analyzed.ResultsHigh concentration angiotensin II with a long-term treatment caused apoptosis and cell viability drop-off in iPSC-CM. Specifically, under a 10-day treatment with 1 mM angiotensin II, the viability of iPSC-CM was reduced by an average of 41% (p=2.073E-08), and the percentage of apoptotic cells was 2.74 times higher than the controls averagely (p=6.248E-12). The data mining of previous RNA-seq data revealed that angiotensin II receptor type I was the major receptor in iPSC-CM. Conclusions: For the first time, our data confirmed the apoptotic effect of angiotensin II to iPSC-CM. The angiotensin II concentrations and exposure time for apoptosis induction were depicted in our study, which provided supports to iPSC-CM as an in vitro model for cardiovascular disease study.

Sign in / Sign up

Export Citation Format

Share Document