scholarly journals Incomplete Assembly of the Dystrophin-Associated Protein Complex in 2D and 3D-Cultured Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

2021 ◽  
Vol 9 ◽  
Author(s):  
Guillaume Gilbert ◽  
Chandan Kadur Nagaraju ◽  
Robin Duelen ◽  
Matthew Amoni ◽  
Pierre Bobin ◽  
...  
Keyword(s):  
Protein Complex ◽  
Cardiac Tissue ◽  
Genetic Diseases ◽  
3D Culture ◽  
Induced Pluripotent Stem Cell ◽  
Mature Adult ◽  
Adult Cardiomyocytes ◽  
Protein Components ◽  
Induced Pluripotent ◽  
2D And 3D

Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) are increasingly used to study genetic diseases on a human background. However, the lack of a fully mature adult cardiomyocyte phenotype of hiPSC-CM may be limiting the scope of these studies. Muscular dystrophies and concomitant cardiomyopathies result from mutations in genes encoding proteins of the dystrophin-associated protein complex (DAPC), which is a multi-protein membrane-spanning complex. We examined the expression of DAPC components in hiPSC-CM, which underwent maturation in 2D and 3D culture protocols. The results were compared with human adult cardiac tissue and isolated cardiomyocytes. We found that similarly to adult cardiomyocytes, hiPSC-CM express dystrophin, in line with previous studies on Duchenne’s disease. β-dystroglycan was also expressed, but, contrary to findings in adult cardiomyocytes, none of the sarcoglycans nor α-dystroglycan were, despite the presence of their mRNA. In conclusion, despite the robust expression of dystrophin, the absence of several other DAPC protein components cautions for reliance on commonly used protocols for hiPSC-CM maturation for functional assessment of the complete DAPC.

Bioengineering approaches to mature induced pluripotent stem cell-derived atrial cardiomyocytes to model atrial fibrillation

2021 ◽  
pp. 153537022110091
Author(s):  
Olivia T Ly ◽  
Grace E Brown ◽  
Yong Duk Han ◽  
Dawood Darbar ◽  
Salman R Khetani
Keyword(s):  
Atrial Fibrillation ◽  
3D Culture ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Pharmacologic Therapy ◽  
Mature Adult ◽  
Culture Techniques ◽  
Atrial Cardiomyocytes ◽  
Induced Pluripotent ◽  
Parenchymal Cells

Induced pluripotent stem cells (iPSCs) serve as a robust platform to model several human arrhythmia syndromes including atrial fibrillation (AF). However, the structural, molecular, functional, and electrophysiological parameters of patient-specific iPSC-derived atrial cardiomyocytes (iPSC-aCMs) do not fully recapitulate the mature phenotype of their human adult counterparts. The use of physiologically inspired microenvironmental cues, such as postnatal factors, metabolic conditioning, extracellular matrix (ECM) modulation, electrical and mechanical stimulation, co-culture with non-parenchymal cells, and 3D culture techniques can help mimic natural atrial development and induce a more mature adult phenotype in iPSC-aCMs. Such advances will not only elucidate the underlying pathophysiological mechanisms of AF, but also identify and assess novel mechanism-based therapies towards supporting a more ‘personalized’ (i.e. patient-specific) approach to pharmacologic therapy of AF.

Use of 3D culture systems to generate human induced pluripotent stem cell-derived [beta]-cells in vitro

2018 ◽  
Author(s):  
Fantuzzi Federica ◽  
Toivonen Sanna ◽  
Schiavo Andrea Alex ◽  
Pachera Nathalie ◽  
Rajaei Bahareh ◽  
...  
Keyword(s):  
Stem Cell ◽  
Beta Cells ◽  
Pluripotent Stem Cell ◽  
3D Culture ◽  
Induced Pluripotent Stem Cell ◽  
Culture Systems ◽  
Induced Pluripotent

Abstract 366: Engineered Human Cardiac Tissue from Skeletal Muscle Derived Cells and Induced Pluripotent Stem Cell Derived Cardiomyocytes

2012 ◽  
Vol 111 (suppl_1) ◽  
Author(s):  
Kimimasa Tobita ◽  
Jason S Tchao ◽  
Jong Kim ◽  
Bo Lin ◽  
Johnny Huard ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Cardiac Tissue ◽  
Troponin I ◽  
Troponin T ◽  
Induced Pluripotent Stem Cell ◽  
Human Cardiac Tissue ◽  
Induced Pluripotent ◽  
Immature Myocardium

We have previously shown that rat skeletal muscle derived stem cells differentiate into an immature cardiomyocyte (CM) phenotype within a 3-dimensional collagen gel engineered cardiac tissue (ECT). Here, we investigated whether human skeletal muscle derived progenitor cells (skMDCs) can differentiate into a CM phenotype within ECT similar to rat skeletal muscle stem cells and compared the human skMDC-ECT properties with ECT from human induced pluripotent stem cell (iPSc) derived CMs. SkMDCs differentiated into a cardiac muscle phenotype within ECT and exhibited spontaneous beating activity as early as culture day 4 and maintained their activity for more than 2 weeks. SkMDC-ECTs stained positive for cardiac specific troponin-T and troponin-I, and were co-localized with fast skeletal muscle myosin heavy chain (sk-fMHC) with a striated muscle pattern similar to fetal myocardium. The iPS-CM-ECTs maintained spontaneous beating activity for more than 2 weeks from ECT construction. iPS-CM stained positive for both cardiac troponin-T and troponin-I, and were also co-localized with sk-fMHC while the striated expression pattern of sk-fMHC was lost similar to post-natal immature myocardium. Connexin-43 protein was expressed in both engineered tissue types, and the expression pattern was similar to immature myocardium. The skMDC-ECT significantly upregulated expression of cardiac-specific genes compared to conventional 2D culture. SkMDC-ECT displayed cardiac muscle like intracellular calcium ion transients. The contractile force measurements demonstrated functional properties of fetal type myocardium in both ECTs. Our results suggest that engineered human cardiac tissue from skeletal muscle progenitor cells mimics developing fetal myocardium while the engineered cardiac tissue from inducible pluripotent stem cell-derived cardiomyocytes mimics post-natal immature myocardium.

Generation of 2.5D lung bud organoid from human induced pluripotent stem cell

2021 ◽  
pp. 1-14
Author(s):  
Xun Xu ◽  
Yan Nie ◽  
Weiwei Wang ◽  
Imran Ullah ◽  
Wing Tai Tung ◽  
...  
Keyword(s):  
Single Cell ◽  
Disease Modeling ◽  
3D Culture ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Homogeneous Distribution ◽  
Cell Seeding ◽  
Differentiation Potential ◽  
Induced Pluripotent ◽  
Defined Culture

Human induced pluripotent stem cells (hiPSCs) are a promising cell source to generate the patient-specific lung organoid given their superior differentiation potential. However, the current 3D cell culture approach is tedious and time-consuming with a low success rate and high batch-to-batch variability. Here, we explored the establishment of lung bud organoids by systematically adjusting the initial confluence levels and homogeneity of cell distribution. The efficiency of single cell seeding and clump seeding was compared. Instead of the traditional 3D culture, we established a 2.5D organoid culture to enable the direct monitoring of the internal structure via microscopy. It was found that the cell confluence and distribution prior to induction were two key parameters, which strongly affected hiPSC differentiation trajectories. Lung bud organoids with positive expression of NKX 2.1, in a single-cell seeding group with homogeneously distributed hiPSCs at 70%confluence (SC_70%_hom) or a clump seeding group with heterogeneously distributed cells at 90%confluence (CL_90%_het), can be observed as early as 9 days post induction. These results suggest that a successful lung bud organoid formation with single-cell seeding of hiPSCs requires a moderate confluence and homogeneous distribution of cells, while high confluence would be a prominent factor to promote the lung organoid formation when seeding hiPSCs as clumps. 2.5D organoids generated with defined culture conditions could become a simple, efficient, and valuable tool facilitating drug screening, disease modeling and personalized medicine.

Abstract 153: Using the Embryonic Heart as an Instructive Template for Cardiac Tissue Engineering

2015 ◽  
Vol 117 (suppl_1) ◽  
Author(s):  
Ivan Batalov ◽  
Quentin Jallerat ◽  
Adam W Feinberg
Keyword(s):  
Tissue Engineering ◽  
Heart Muscle ◽  
Cardiac Tissue ◽  
Microcontact Printing ◽  
Induced Pluripotent Stem Cell ◽  
Cardiac Tissue Engineering ◽  
Embryonic Heart ◽  
Embryonic Chick ◽  
Tissue Engineering Scaffolds ◽  
Induced Pluripotent

The engineering of highly aligned cardiomyocytes into functional heart muscle remains a primary challenge in cardiac tissue engineering. Researchers have shown that micropatterned topography and chemistry as well as mechanical and electrical gradients are all effective at inducing some degree of alignment. However, which approach works best in terms of electromechanical function of the engineered cardiac muscle is still an active area of research. Because formation of new heart muscle in mammals primarily occurs during cardiogenesis, we asked whether the embryonic heart could be used as an instructive template for the design of more effective cardiac tissue engineering scaffolds. Specifically, we hypothesized that micropatterns of fibronectin based on fibronectin fibril size and architecture in embryonic myocardium could improve cardiomyocyte alignment relative to 20 μm wide, 20 μm spaced fibronectin lines, a control pattern used widely in the literature. To test this, we first imaged the fibronectin matrix in the ventricles of day-5 embryonic chick hearts and imaged this in 3D using a multiphoton microscope. This fibronectin structure was then converted into a photomask for photolithography and subsequent patterning of fibronectin onto cover slips using microcontact printing. Samples with the biomimetic patterns or control patterns were seeded with embryonic chick cardiomyocytes, cultured for 3 days and then stained and imaged to visualize the myofibrils. Image analysis to quantify alignment showed that the ability of the biomimetic pattern to induce cardiomyocyte alignment increased with cell density, suggesting that cell-cell interactions play an important role in the formation of aligned embryonic myocardium. Disruption of the cadherins junctions using blocking antibodies confirmed this conclusion. In the future we will use human induced pluripotent stem cell-derived cardiomyocytes to engineer more clinically-relevant human heart muscle and analyze electromechanical function of the tissues including contractile force and action potential propagation.

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