scholarly journals Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro

2017 ◽  
Vol 2017 ◽  
pp. 1-18 ◽  
Author(s):  
Divya S. Varghese ◽  
Shama Parween ◽  
Mustafa T. Ardah ◽  
Bright Starling Emerald ◽  
Suraiya A. Ansari
Keyword(s):  
Cell Viability ◽  
Disease Modeling ◽  
Culture Media ◽  
Embryonic Stem ◽  
Toxicity Testing ◽  
Cell Types ◽  
Specific Cell ◽  
Directed Differentiation ◽  
Embryonic Neurogenesis

Human embryonic stem cells (hESCs) are being used extensively in array of studies to understand different mechanisms such as early human embryogenesis, drug toxicity testing, disease modeling, and cell replacement therapy. The protocols for the directed differentiation of hESCs towards specific cell types often require long-term cell cultures. To avoid bacterial contamination, these protocols include addition of antibiotics such as pen-strep and gentamicin. Although aminoglycosides, streptomycin, and gentamicin have been shown to cause cytotoxicity in various animal models, the effect of these antibiotics on hESCs is not clear. In this study, we found that antibiotics, pen-strep, and gentamicin did not affect hESC cell viability or expression of pluripotency markers. However, during directed differentiation towards neural and hepatic fate, significant cell death was noted through the activation of caspase cascade. Also, the expression of neural progenitor markers Pax6, Emx2, Otx2, and Pou3f2 was significantly reduced suggesting that gentamicin may adversely affect early embryonic neurogenesis whereas no effect was seen on the expression of endoderm or hepatic markers during differentiation. Our results suggest that the use of antibiotics in cell culture media for the maintenance and differentiation of hESCs needs thorough investigation before use to avoid erroneous results.

scholarly journals An efficient method to generate kidney organoids at the air-liquid interface

2021 ◽  
Vol 8 (2) ◽  
pp. e150
Author(s):  
Ashwani Kumar Gupta ◽  
David Z. Ivancic ◽  
Bilal A. Naved ◽  
Jason A. Wertheim ◽  
Leif Oxburgh
Keyword(s):  
Disease Modeling ◽  
Embryonic Stem ◽  
Cell Types ◽  
Culture Conditions ◽  
Specific Cell ◽  
Directed Differentiation ◽  
Chemical Screening ◽  
Air Liquid Interface ◽  
Kidney Organoids

The prevalence of kidney dysfunction continues to increase worldwide, driving the need to develop transplantable renal tissues. The kidney develops from four major renal progenitor populations: nephron epithelial, ureteric epithelial, interstitial and endothelial progenitors. Methods have been developed to generate kidney organoids but few or dispersed tubular clusters within the organoids hamper its use in regenerative applications. Here, we describe a detailed protocol of asynchronous mixing of kidney progenitors using organotypic culture conditions to generate kidney organoids tightly packed with tubular clusters and major renal structures including endothelial network and functional proximal tubules. This protocol provides guidance in the culture of human embryonic stem cells from a National Institute of Health-approved line and their directed differentiation into kidney organoids. Our 18-day protocol provides a rapid method to generate kidney organoids that facilitate the study of different nephrological events including in vitro tissue development, disease modeling and chemical screening. However, further studies are required to optimize the protocol to generate additional renal-specific cell types, interconnected nephron segments and physiologically functional renal tissues.

scholarly journals Pancreatic Progenitors and Organoids as a Prerequisite to Model Pancreatic Diseases and Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-11 ◽  
Author(s):  
Meike Hohwieler ◽  
Martin Müller ◽  
Pierre-Olivier Frappart ◽  
Sandra Heller
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Disease Modeling ◽  
Embryonic Stem ◽  
Cell Types ◽  
Genetically Engineered ◽  
Hereditary Diseases ◽  
Pancreatic Diseases ◽  
Pancreatic Progenitors

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are characterized by their unique capacity to stepwise differentiate towards any particular cell type in an adult organism. Pluripotent stem cells provide a beneficial platform to model hereditary diseases and even cancer development. While the incidence of pancreatic diseases such as diabetes and pancreatitis is increasing, the understanding of the underlying pathogenesis of particular diseases remains limited. Only a few recent publications have contributed to the characterization of human pancreatic development in the fetal stage. Hence, most knowledge of pancreatic specification is based on murine embryology. Optimizing and understanding current in vitro protocols for pancreatic differentiation of ESCs and iPSCs constitutes a prerequisite to generate functional pancreatic cells for better disease modeling and drug discovery. Moreover, human pancreatic organoids derived from pluripotent stem cells, organ-restricted stem cells, and tumor samples provide a powerful technology to model carcinogenesis and hereditary diseases independent of genetically engineered mouse models. Herein, we summarize recent advances in directed differentiation of pancreatic organoids comprising endocrine cell types. Beyond that, we illustrate up-and-coming applications for organoid-based platforms.

scholarly journals Human Organoids for Predictive Toxicology Research and Drug Development

2021 ◽  
Vol 12 ◽  
Author(s):  
Toshikatsu Matsui ◽  
Tadahiro Shinozawa
Keyword(s):  
Stem Cells ◽  
Drug Development ◽  
Disease Modeling ◽  
Cell Types ◽  
Underlying Disease ◽  
Future Research ◽  
Specific Cell ◽  
Predictive Toxicology

Organoids are three-dimensional structures fabricated in vitro from pluripotent stem cells or adult tissue stem cells via a process of self-organization that results in the formation of organ-specific cell types. Human organoids are expected to mimic complex microenvironments and many of the in vivo physiological functions of relevant tissues, thus filling the translational gap between animals and humans and increasing our understanding of the mechanisms underlying disease and developmental processes. In the last decade, organoid research has attracted increasing attention in areas such as disease modeling, drug development, regenerative medicine, toxicology research, and personalized medicine. In particular, in the field of toxicology, where there are various traditional models, human organoids are expected to blaze a new path in future research by overcoming the current limitations, such as those related to differences in drug responses among species. Here, we discuss the potential usefulness, limitations, and future prospects of human liver, heart, kidney, gut, and brain organoids from the viewpoints of predictive toxicology research and drug development, providing cutting edge information on their fabrication methods and functional characteristics.

scholarly journals The Production and Directed Differentiation of Human Embryonic Stem Cells

2006 ◽  
Vol 27 (2) ◽  
pp. 208-219 ◽  
Author(s):  
Alan Trounson
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Regenerative Medicine ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Directed Differentiation ◽  
Hesc Lines

Human embryonic stem cells (hESCs) are being rapidly produced from chromosomally euploid, aneuploid, and mutant human embryos that are available from in vitro fertilization clinics treating patients for infertility or preimplantation genetic diagnosis. These hESC lines are an important resource for functional genomics, drug screening, and, perhaps eventually, cell and gene therapy. The methods for deriving hESCs are well established and repeatable and are relatively successful with a ratio of 1:10 to 1:2 new hESC lines produced from 4- to 8-d-old morula and blastocysts and from isolated inner cell mass cell clusters of human blastocysts. The hESCs can be formed and maintained on human somatic cells in humanized serum-free culture conditions and for several passages in cell-free culture systems. The hESCs can be transfected with DNA constructs. Their gene expression profiles are being described and immunological characteristics determined. They may be grown indefinitely in vitro while maintaining their original karyotype and epigenetic status, but this needs to be confirmed from time to time in long-term cultures. hESCs spontaneously differentiate in the absence of the appropriate cell feeder layer, when overgrown in culture and when isolated from the ESC colony. All three major embryonic lineages are produced in differentiating flat attachment cultures and unattached embryoid bodies. Cell progenitors of interest can be identified by markers, expression of reporter genes, and characteristic morphology, and the cells thereafter enriched for progenitor types and further culture to more mature cell types. Directed differentiation systems are well developed for ectodermal pathways that result in neural and glial cells and the mesendodermal pathway for cardiac muscle cells and many other cell types including hematopoietic progenitors and endothelial cells. Directed differentiation into endoderm has been more difficult to achieve, perhaps because of the lack of markers of early progenitors in this lineage. There are reports of enriched cultures of keratinocytes, pigmented retinal epithelium, neural crest cells and motor neurons, hepatic progenitors, and cells that have some markers of gut tissue and pancreatic islet-like cells. The prospects for use of hESC derivatives in regenerative medicine are significant, and there is much optimism for their potential contributions to human regenerative medicine.

Potential benefits of cell cloning for human medicine

1998 ◽  
Vol 10 (1) ◽  
pp. 121 ◽  
Author(s):  
A. Trounson ◽  
M. Pera
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Somatic Cell ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Specific Cell ◽  
Somatic Cell Nucleus ◽  
Potential Benefits

The successful cloning of a mammal from an adult somatic cell nucleus opens new avenues for major advances in reproductive medicine, biotechnology and cellular-based transplantation therapies for degenerative diseases. At the same time, this breakthrough has generated much heated discussion concerning the ethics of cloning. Twinning is a form of cloning, and there are instances in clinical assisted reproduction in which the deliberate formation of twins by embryo dissection would seem ethically acceptable. Nuclear transfer technology might facilitate the derivation of human embryonic stem cells, capable of differentiation into a wide variety of somatic cell lineages. Directed differentiation of human embryonic stem cells into specific cell types in vitro could provide a universal source of cells for transplantation therapy. The potential benefits of therapeutics based on cloning technologies are considerable, and hasty legislation to ban all such procedures could block progress in critical arenas of biomedical research

scholarly journals Modeling Kidney Disease with iPS Cells

2015 ◽  
Vol 10s1 ◽  
pp. BMI.S20054 ◽  
Author(s):  
Benjamin S. Freedman
Keyword(s):  
Kidney Disease ◽  
Disease Modeling ◽  
Embryonic Stem ◽  
Clinical Information ◽  
Cell Types ◽  
Directed Differentiation ◽  
Design Strategies ◽  
Induced Pluripotent ◽  
Laboratory Models ◽  
Kidney Organoids

Induced pluripotent stem cells (iPSCs) are somatic cells that have been transcriptionally reprogrammed to an embryonic stem cell (ESC)-like state. iPSCs are a renewable source of diverse somatic cell types and tissues matching the original patient, including nephron-like kidney organoids. iPSCs have been derived representing several kidney disorders, such as ADPKD, ARPKD, Alport syndrome, and lupus nephritis, with the goals of generating replacement tissue and ‘disease in a dish’ laboratory models. Cellular defects in iPSCs and derived kidney organoids provide functional, personalized biomarkers, which can be correlated with genetic and clinical information. In proof of principle, disease-specific phenotypes have been described in iPSCs and ESCs with mutations linked to polycystic kidney disease or focal segmental glomerulosclerosis. In addition, these cells can be used to model nephrotoxic chemical injury. Recent advances in directed differentiation and CRISPR genome editing enable more specific iPSC models and present new possibilities for diagnostics, disease modeling, therapeutic screens, and tissue regeneration using human cells. This review outlines growth opportunities and design strategies for this rapidly expanding and evolving field.

scholarly journals De novo generation of HSCs from somatic and pluripotent stem cell sources

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2641-2648 ◽  
Author(s):  
Linda T. Vo ◽  
George Q. Daley
Keyword(s):  
Stem Cells ◽  
Disease Modeling ◽  
De Novo ◽  
Cell Types ◽  
Direct Conversion ◽  
Cellular Reprogramming ◽  
Directed Differentiation ◽  
Hematopoietic Stem ◽  
Cell Sources

Abstract Generating human hematopoietic stem cells (HSCs) from autologous tissues, when coupled with genome editing technologies, is a promising approach for cellular transplantation therapy and for in vitro disease modeling, drug discovery, and toxicology studies. Human pluripotent stem cells (hPSCs) represent a potentially inexhaustible supply of autologous tissue; however, to date, directed differentiation from hPSCs has yielded hematopoietic cells that lack robust and sustained multilineage potential. Cellular reprogramming technologies represent an alternative platform for the de novo generation of HSCs via direct conversion from heterologous cell types. In this review, we discuss the latest advancements in HSC generation by directed differentiation from hPSCs or direct conversion from somatic cells, and highlight their applications in research and prospects for therapy.

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

2018 ◽  
Vol 18 (4) ◽  
pp. 246-255 ◽  
Author(s):  
Lara Termini ◽  
Enrique Boccardo
Keyword(s):  
Three Dimensional ◽  
Cell Types ◽  
Experimental Models ◽  
Biological Research ◽  
Specific Gene ◽  
Specific Cell ◽  
Organotypic Cultures ◽  
Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

Nephrotoxicity Testing In Vitro: The Current Situation

1997 ◽  
Vol 25 (5) ◽  
pp. 497-503
Author(s):  
Jean-Paul Morin ◽  
Marc E. De Broe ◽  
Walter Pfaller ◽  
Gabriele Schmuck
Keyword(s):  
Proximal Tubule ◽  
Culture Media ◽  
Task Force ◽  
Primary Cultures ◽  
Phenotypic Expression ◽  
Transepithelial Transport ◽  
Specific Cell ◽  
Proximal Tubule Cells ◽  
Tubule Cells

An ECVAM task force on nephrotoxicity has been established to advise, in particular, on the follow-up to recommendations made in the ECVAM workshop report on nephrotoxicity testing in vitro. Since this workshop was held, in 1994, there have been several improvements in the techniques used. For example, the duration of renal slice viability, and the maintenance of functional activities in slices, have been improved by using dynamic incubation systems with higher oxygen tensions and more-appropriate cell culture media. Highly differentiated primary cultures of pig, human and rabbit proximal tubule cells have been established by using specific cell isolation procedures and/or selective culture media. To date, the most comparable phenotypic expression and transepithelial transport capacities to proximal tubules in vivo have been obtained with primary cultures of rabbit proximal tubule cells which are grown on bicompartmental supports; in this system, transepithelial substrate gradients are generated and the transepithelial transport of both organic anions and cations is highly active. This in vitro system has been selected by ECVAM for further evaluation and prevalidation. Industrial needs in the area of nephrotoxicity testing have been identified, and recommendations are made at the end of this report concerning possible future initiatives.

scholarly journals Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells

Nanomaterials ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 1929
Author(s):  
Tereza Cervena ◽  
Andrea Rossnerova ◽  
Tana Zavodna ◽  
Jitka Sikorova ◽  
Kristyna Vrbova ◽  
...  
Keyword(s):  
Cytochalasin B ◽  
Culture Media ◽  
Methodological Approach ◽  
Micronucleus Assay ◽  
Toxicity Testing ◽  
In Vitro Toxicity ◽  
Testing Strategies ◽  
Transmission Electron ◽  
The Impact

The evaluation of the frequency of micronuclei (MN) is a broadly utilised approach in in vitro toxicity testing. Nevertheless, the specific properties of nanomaterials (NMs) give rise to concerns regarding the optimal methodological variants of the MN assay. In bronchial epithelial cells (BEAS-2B), we tested the genotoxicity of five types of NMs (TiO2: NM101, NM103; SiO2: NM200; Ag: NM300K, NM302) using four variants of MN protocols, differing in the time of exposure and the application of cytochalasin-B combined with the simultaneous and delayed co-treatment with NMs. Using transmission electron microscopy, we evaluated the impact of cytochalasin-B on the transport of NMs into the cells. To assess the behaviour of NMs in a culture media for individual testing conditions, we used dynamic light scattering measurement. The presence of NMs in the cells, their intracellular aggregation and dispersion properties were comparable when tests with or without cytochalasin-B were performed. The genotoxic potential of various TiO2 and Ag particles differed (NM101 < NM103 and NM302 < NM300K, respectively). The application of cytochalasin-B tended to increase the percentage of aberrant cells. In conclusion, the comparison of the testing strategies revealed that the level of DNA damage induced by NMs is affected by the selected methodological approach. This fact should be considered in the interpretation of the results of genotoxicity tests.

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