RNAseq Analysis of Isogenic Human Induced Pluripotent Stem Cell Lines Reveals Differential Gene Expression Patterns during Differentiation of Human Heart Organoids Due to a Patient-Specific TBX5 Mutation
Harald LahmBernd H. NorthoffMarina ReinschAkio MantaniIrina NebElda DzilicS. DopplerRüdiger LangeLesca M. HoldtArne HansenMarkus KraneMartina Dreßen
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Human pluripotent stem cells have the potential to change the way in which human diseases are cured. Clinical-grade human embryonic stem cells and human induced pluripotent stem cells have to be created according to current good manufacturing practices and regulations. Quality and safety must be of the highest importance when humans’ lives are at stake. With the rising number of clinical trials, there is a need for a consensus on hPSCs characterization. Here, we summarize mandatory and ′for information only′ characterization methods with release criteria for the establishment of clinical-grade hPSC lines.
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Purpose: Two-dimensional (2D)-based cell culture systems, limited by their inherent heterogeneity and scalability, are a bottleneck in the production of high-quality cells for downstream biomedical applications. Finding the optimal conditions for large-scale stem cell culture while maintaining good cellular status is challenging. The aim of this study was to assess the effects of three-dimensional (3D) culture on the viability, proliferation, self-renewal, and differentiation of human induced pluripotent stem cells (IPSCs). Patients and Methods: Various culture conditions were evaluated to determine the optimal conditions to maintain the viability and proliferation of human IPSCs in a 3D environment: static versus dynamic culture, type of adhesion protein added to alginate (Matrigel™ versus gelatin), and the addition of Y-27632t on long-term 3D culture. The proliferation ability of the cells was evaluated via the MTS proliferation assay; the expression levels of the pluripotency markers Nanog and Oct3/4, PAX6 as an ectoderm marker, and laminin-5 and fibronectin as markers of extracellular matrix synthesis were assessed; and HIF1α and HIF2α levels were measured using quantitative reverse transcription polymerase chain reaction. Results: Using a high-aspect-ratio vessel bioreactor with a gentle, low-sheer, and low-turbulence environment with sufficient oxygenation and effective mass transfer of nutrients and waste, we verified its ability to promote cell proliferation and self-renewal. The findings showed that human IPSCs have the ability to maintain pluripotency in a feeder-free system and by inhibiting ROCK signaling and using hypoxia to improve single-cell viability in 3D culture. Furthermore, these cells demonstrated increased self-renewal and proliferation when inoculated as single cells in 3D alginate beads by adding RI during the culture period. Conclusion: Dynamic 3D culture is desirable for the large-scale expansion of undifferentiated human IPSCs. Keywords: stem cell culture, large-scale expansion, HARV bioreactor, self-renewal
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Induced pluripotent stem cells(iPSCs) not only have the ability to proliferate indefinitely,but can filter according to different needs of particular phenotypes.Furthermore,i PSCs could be directionally induced to produce red blood cells(RBC) and provide a new source for RBC transfusion.However,the application of this technology to the clinical transfusion still needs optimizations on several key steps,such as the selection of types of cells which produce i PSCs,the improvement on reprogramming methods that could be applied to guarantee the safety of clinical applications,and the optimization of erythrocyte differentiation process.
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Introduction: In murine myocardial injury model, the favorable effects of iCMs transplantation are not completely understood. Exosomes secreted from transplanted cells may mediate beneficial effect...
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Since the first studies of human embryonic stem cells (hESCs) and, more recently, human induced pluripotent stem cells (hiPSCs), the stem-cell field has been abuzz with the promise that these pluripotent populations will one day be a powerful therapeutic tool. Although it has been proposed that hiPSCs will supersede hESCs with respect to their research and/or clinical potential because of the ease of their derivation and the ability to create immunologically matched iPSCs for each individual patient, recent evidence suggests that iPSCs in fact have several underappreciated characteristics that might mean they are less suitable for clinical application. Continuing research is revealing the similarities, differences and deficiencies of various pluripotent stem-cell populations, and suggests that many years will pass before the clinical utility of hESCs and hiPSCs is realized. There are a plethora of ethical, logistical and technical roadblocks on the route to the clinical application of pluripotent stem cells, particularly of iPSCs. In this Essay, we discuss what we believe are important issues that should be considered when attempting to bring hiPSC-based technology to the clinic.
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In this protocol we describe the differentiation of human induced pluripotent stem cells (hiPSCs) into human midbrain-like organoids (hMLOs). This protocol has been developed based from several published protocols.
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Research of genetic cardiovascular diseases has lacked of good disease models because rodents, which are primarily used, differ greatly from humans. The ability to derive human induced pluripotent stem cells (hiPSCs) from patients carrying inherited cardiac diseases has revolutionized research in the cardiovascular field. The aim for this chapter is to review the current hiPSC reprogramming methods and methods for differentiating human pluripotent stem cells (hPSCs) into cardiomyocytes. The chapter focuses on the published hiPSC models for hypertrophic cardiomyopathy (HCM) and discusses the challenges related to modeling this interesting disease using hiPSC technology.
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Mature liver organoids are promising cell sources for research to understand the pathology underlying a variety of conditions affecting the liver, including end-stage chronic liver disease. Although several methods exist for the differentiation of mature hepatic organoids derived from human induced pluripotent stem cells (hiPSCs), organoid generation can fail due to various experimental culture conditions. Therefore, we established a standard operating protocol for generating mature and expandable hepatic organoids derived from hiPSCs, and we made the starting materials available to facilitate the wide use of the protocol.
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