scholarly journals Cellular Reprogramming Employing Recombinant Sox2 Protein

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Marc Thier ◽  
Bernhard Münst ◽  
Stephanie Mielke ◽  
Frank Edenhofer
Keyword(s):  
Protein Delivery ◽  
Disease Modeling ◽  
Ips Cells ◽  
Cellular Reprogramming ◽  
Protein Stabilization ◽  
Biologically Active ◽  
Patient Specific ◽  
Tat Protein ◽  
Serum Replacement ◽  
Induced Pluripotent

Induced pluripotent stem (iPS) cells represent an attractive option for the derivation of patient-specific pluripotent cells for cell replacement therapies as well as disease modeling. To become clinically meaningful, safe iPS cells need to be generated exhibiting no permanent genetic modifications that are caused by viral integrations of the reprogramming transgenes. Recently, various experimental strategies have been applied to accomplish transgene-free derivation of iPS cells, including the use of nonintegrating viruses, episomal expression, or excision of transgenes after reprogramming by site-specific recombinases or transposases. A straightforward approach to induce reprogramming factors is the direct delivery of either synthetic mRNA or biologically active proteins. We previously reported the generation of cell-permeant versions of Oct4 (Oct4-TAT) and Sox2 (Sox2-TAT) proteins and showed that Oct4-TAT is reprogramming-competent, that is, it can substitute for Oct4-encoding virus. Here, we explore conditions for enhanced Sox2-TAT protein stabilization and functional delivery into somatic cells. We show that cell-permeant Sox2 protein can be stabilized by lipid-rich albumin supplements in serum replacement or low-serum-supplemented media. Employing optimized conditions for protein delivery, we demonstrate that Sox2-TAT protein is able to substitute for viral Sox2. Sox2-piPS cells express pluripotency-associated markers and differentiate into all three germ layers.

scholarly journals The Promise of Human Induced Pluripotent Stem Cells in Dental Research

2012 ◽  
Vol 2012 ◽  
pp. 1-10 ◽  
Author(s):  
Thekkeparambil Chandrabose Srijaya ◽  
Padmaja Jayaprasad Pradeep ◽  
Rosnah Binti Zain ◽  
Sabri Musa ◽  
Noor Hayaty Abu Kasim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Patient Specific ◽  
Dental Research ◽  
Cell Based Therapy ◽  
Induced Pluripotent ◽  
Disease Specific

Induced pluripotent stem cell-based therapy for treating genetic disorders has become an interesting field of research in recent years. However, there is a paucity of information regarding the applicability of induced pluripotent stem cells in dental research. Recent advances in the use of induced pluripotent stem cells have the potential for developing disease-specific iPSC linesin vitrofrom patients. Indeed, this has provided a perfect cell source for disease modeling and a better understanding of genetic aberrations, pathogenicity, and drug screening. In this paper, we will summarize the recent progress of the disease-specific iPSC development for various human diseases and try to evaluate the possibility of application of iPS technology in dentistry, including its capacity for reprogramming some genetic orodental diseases. In addition to the easy availability and suitability of dental stem cells, the approach of generating patient-specific pluripotent stem cells will undoubtedly benefit patients suffering from orodental disorders.

scholarly journals Machine Learning Approach to Automated Quality Identification of Human Induced Pluripotent Stem Cell Colony Images

2016 ◽  
Vol 2016 ◽  
pp. 1-15 ◽  
Author(s):  
Henry Joutsijoki ◽  
Markus Haponen ◽  
Jyrki Rasku ◽  
Katriina Aalto-Setälä ◽  
Martti Juhola
Keyword(s):  
Machine Learning ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Support Vector ◽  
Ips Cell ◽  
Cell Technology ◽  
Induced Pluripotent

The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient’s cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using ak-NN classifier showing improved accuracy compared to earlier studies.

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.

scholarly journals The Application of Induced Pluripotent Stem Cells in Pathogenesis Study and Gene Therapy for Vascular Disorders: Current Progress and Future Challenges

2019 ◽  
Vol 2019 ◽  
pp. 1-9 ◽  
Author(s):  
Guang-Yin Peng ◽  
Yang Lin ◽  
Jing-Jing Li ◽  
Ying Wang ◽  
Hao-Yue Huang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Vascular Diseases ◽  
Patient Specific ◽  
High Morbidity ◽  
Vascular Disorders ◽  
Induced Pluripotent

Vascular disorders are complex diseases with high morbidity and mortality. Among them, the dilated macrovascular diseases (MVD), such as aortic aneurysm and aortic dissection, have presented a huge threat to human health. The pathogenesis of vascular diseases is mostly associated with property alteration of vascular endothelial cells (VECs) and vascular smooth muscle cells (VSMCs). Studies have confirmed that induced pluripotent stem cells (iPSCs) can be proliferated and differentiated into other somatic cells, such as VECs and VSMCs. And patient-specific cells could provide detailed human-associated information in regard to pathogenesis or drug responses. In addition, differentiated ECs from iPSC have been widely used in disease modeling as a cell therapy. In this review, we mainly discussed the application of hiPSCs in investigating the pathological mechanism of different inherited vascular diseases and provide a comprehensive understanding of hiPSCs in the field of clinical diagnosis and gene therapy.

scholarly journals “Betwixt Mine Eye and Heart a League Is Took”: The Progress of Induced Pluripotent Stem-Cell-Based Models of Dystrophin-Associated Cardiomyopathy

2020 ◽  
Vol 21 (19) ◽  
pp. 6997 ◽  
Author(s):  
Davide Rovina ◽  
Elisa Castiglioni ◽  
Francesco Niro ◽  
Sara Mallia ◽  
Giulio Pompilio ◽  
...  
Keyword(s):  
Stem Cell ◽  
Muscular Dystrophy ◽  
Pluripotent Stem Cell ◽  
Disease Modeling ◽  
Disease Model ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Great Promise ◽  
Cord Injury ◽  
Induced Pluripotent

The ultimate goal of precision disease modeling is to artificially recreate the disease of affected people in a highly controllable and adaptable external environment. This field has rapidly advanced which is evident from the application of patient-specific pluripotent stem-cell-derived precision therapies in numerous clinical trials aimed at a diverse set of diseases such as macular degeneration, heart disease, spinal cord injury, graft-versus-host disease, and muscular dystrophy. Despite the existence of semi-adequate treatments for tempering skeletal muscle degeneration in dystrophic patients, nonischemic cardiomyopathy remains one of the primary causes of death. Therefore, cardiovascular cells derived from muscular dystrophy patients’ induced pluripotent stem cells are well suited to mimic dystrophin-associated cardiomyopathy and hold great promise for the development of future fully effective therapies. The purpose of this article is to convey the realities of employing precision disease models of dystrophin-associated cardiomyopathy. This is achieved by discussing, as suggested in the title echoing William Shakespeare’s words, the settlements (or “leagues”) made by researchers to manage the constraints (“betwixt mine eye and heart”) distancing them from achieving a perfect precision disease model.

scholarly journals Disease Modeling of Mitochondrial Cardiomyopathy Using Patient-Specific Induced Pluripotent Stem Cells

Biology ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 981
Author(s):  
Takeshi Tokuyama ◽  
Razan Elfadil Ahmed ◽  
Nawin Chanthra ◽  
Tatsuya Anzai ◽  
Hideki Uosaki
Keyword(s):  
Pluripotent Stem Cells ◽  
Genetic Variants ◽  
Molecular Mechanisms ◽  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Drug Efficacy ◽  
Patient Specific ◽  
Experimental Platform ◽  
Mitochondrial Cardiomyopathy ◽  
Induced Pluripotent

Mitochondrial cardiomyopathy (MCM) is characterized as an oxidative phosphorylation disorder of the heart. More than 100 genetic variants in nuclear or mitochondrial DNA have been associated with MCM. However, the underlying molecular mechanisms linking genetic variants to MCM are not fully understood due to the lack of appropriate cellular and animal models. Patient-specific induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) provide an attractive experimental platform for modeling cardiovascular diseases and predicting drug efficacy to such diseases. Here we introduce the pathological and therapeutic studies of MCM using iPSC-CMs and discuss the questions and latest strategies for research using iPSC-CMs.

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 Mutation-specific differences in arrhythmias and drug responses in CPVT patients: simultaneous patch clamp and video imaging of iPSC derived cardiomyocytes

2019 ◽  
Vol 47 (2) ◽  
pp. 1067-1077 ◽  
Author(s):  
R. P. Pölönen ◽  
H. Swan ◽  
K. Aalto-Setälä
Keyword(s):  
Disease Modeling ◽  
Induced Pluripotent Stem Cell ◽  
Polymorphic Ventricular Tachycardia ◽  
Patient Specific ◽  
Electrophysiological Properties ◽  
Electrophysiological Measurements ◽  
Skin Biopsies ◽  
Induced Pluripotent ◽  
Almost All ◽  
And Control

AbstractCatecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease characterized by arrhythmias under adrenergic stress. Mutations in the cardiac ryanodine receptor (RYR2) are the leading cause for CPVT. We characterized electrophysiological properties of CPVT patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying different mutations in RYR2 and evaluated effects of carvedilol and flecainide on action potential (AP) and contractile properties of hiPSC-CMs. iPSC-CMs were generated from skin biopsies of CPVT patients carrying exon 3 deletion (E3D) and L4115F mutation in RYR2. APs and contractile movement were recorded simultaneously from the same hiPSC-CMs. Differences in AP properties of ventricular like CMs were seen in CPVT and control CMs: APD90 of both E3D (n = 20) and L4115F (n = 25) CPVT CMs was shorter than in control CMs (n = 15). E3D-CPVT CMs had shortest AP duration, lowest AP amplitude, upstroke velocity and more depolarized diastolic potential than controls. Adrenaline had positive and carvedilol and flecainide negative chronotropic effect in all hiPSC CMs. CPVT CMs had increased amount of delayed after depolarizations (DADs) and early after depolarizations (EADs) after adrenaline exposure. E3D CPVT CMs had the most DADs, EADs, and tachyarrhythmia. Discordant negatively coupled alternans was seen in L4115F CPVT CMs. Carvedilol cured almost all arrhythmias in L4115F CPVT CMs. Both drugs decreased contraction amplitude in all hiPSC CMs. E3D CPVT CMs have electrophysiological properties, which render them more prone to arrhythmias. iPSC-CMs provide a unique platform for disease modeling and drug screening for CPVT. Combining electrophysiological measurements, we can gain deeper insight into mechanisms of arrhythmias.

Telomere Elongation in Dyskeratosis Congenita Induced Pluripotent Stem Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 497-497
Author(s):  
Suneet Agarwal ◽  
Yuin-Han Loh ◽  
Erin M McLoughlin ◽  
Junjiu Huang ◽  
In-Hyun Park ◽  
...  
Keyword(s):  
Telomere Length ◽  
Autosomal Dominant ◽  
Somatic Cells ◽  
Ips Cells ◽  
Dyskeratosis Congenita ◽  
Telomere Maintenance ◽  
Patient Specific ◽  
Self Renewal ◽  
Induced Pluripotent

Abstract Abstract 497 Patients with dyskeratosis congenita (DC), a disorder of telomere maintenance, suffer premature degeneration of multiple tissues. Bone marrow failure is the principal cause of mortality, and allogeneic stem cell transplantation is limited by increased treatment-related mortality. Somatic cells can be reprogrammed using defined genetic and chemical factors, yielding “induced pluripotent stem” (iPS) cell lines which have the capacity to differentiate into any tissue. Patient-specific iPS cells therefore hold promise as therapeutic agents and disease models for human degenerative disorders like DC. A cardinal feature of iPS cells is acquisition of indefinite self-renewal capacity, and we have found that telomere length is increased in human iPS cells relative to the normal primary somatic cells from which they are derived. Here we investigated whether defects in telomerase function would limit derivation or self-renewal of iPS cells from patients with DC. We reprogrammed primary fibroblasts from patients with X-linked and autosomal dominant DC, caused by mutations in the genes encoding dyskerin and telomerase RNA component (TERC), respectively. We were able to establish multiple DC-specific iPS lines showing all hallmarks of pluripotency, including the formation of hematopoietic progenitors in vitro. Unexpectedly, DC-specific iPS cells were able to sustain continual proliferation in vitro, in contrast to the premature senescence displayed by the DC fibroblasts. Although early passage DC iPS cells had shorter telomeres than donor fibroblasts, we found that telomere length in DC iPS cells increased with continued passage in culture. To explain this finding, we discovered that steady state levels of TERC, which are critically limiting in several forms of DC, are upregulated in normal and DC iPS cells. We found that TERC upregulation is a feature of the pluripotent state, that the TERC locus is a target of pluripotency-associated transcription factors, and that transcriptional silencing accompanies a 3' deletion at the TERC locus in autosomal dominant DC. Our results demonstrate that reprogramming restores self-renewal capacity in DC cells despite genetic lesions affecting telomerase, and suggest that strategies to enhance endogenous TERC expression may be feasible and therapeutically beneficial in DC patients. The studies demonstrate the value of patient-specific iPS cells for basic and translational discovery, and further the rationale for autologous iPS based cellular therapy of genetic hematologic disorders. Disclosures: Daley: MPM Capital: Consultancy; Solasia: Consultancy; Epizyme: Consultancy; iPierian: Consultancy, Equity Ownership.

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