A look into retinal organoids: methods, analytical techniques, and applications

AbstractInherited retinal diseases (IRDs) cause progressive loss of light-sensitive photoreceptors in the eye and can lead to blindness. Gene-based therapies for IRDs have shown remarkable progress in the past decade, but the vast majority of forms remain untreatable. In the era of personalised medicine, induced pluripotent stem cells (iPSCs) emerge as a valuable system for cell replacement and to model IRD because they retain the specific patient genome and can differentiate into any adult cell type. Three-dimensional (3D) iPSCs-derived retina-like tissue called retinal organoid contains all major retina-specific cell types: amacrine, bipolar, horizontal, retinal ganglion cells, Müller glia, as well as rod and cone photoreceptors. Here, we describe the main applications of retinal organoids and provide a comprehensive overview of the state-of-art analysis methods that apply to this model system. Finally, we will discuss the outlook for improvements that would bring the cellular model a step closer to become an established system in research and treatment development of IRDs.

Download Full-text

Modelling inherited cardiac disease using human induced pluripotent stem cell-derived cardiomyocytes: progress, pitfalls, and potential

Cardiovascular Research ◽

10.1093/cvr/cvy208 ◽

2018 ◽

Vol 114 (14) ◽

pp. 1828-1842 ◽

Cited By ~ 10

Author(s):

Alain van Mil ◽

Geerthe Margriet Balk ◽

Klaus Neef ◽

Jan Willem Buikema ◽

Folkert W Asselbergs ◽

...

Keyword(s):

Heart Disease ◽

Induced Pluripotent Stem Cell ◽

Cell Types ◽

Cardiac Cells ◽

Patient Specific ◽

Specific Cell ◽

Comprehensive Overview ◽

Cellular Processes ◽

Ipsc Generation ◽

Induced Pluripotent

Abstract In the past few years, the use of specific cell types derived from induced pluripotent stem cells (iPSCs) has developed into a powerful approach to investigate the cellular pathophysiology of numerous diseases. Despite advances in therapy, heart disease continues to be one of the leading causes of death in the developed world. A major difficulty in unravelling the underlying cellular processes of heart disease is the extremely limited availability of viable human cardiac cells reflecting the pathological phenotype of the disease at various stages. Thus, the development of methods for directed differentiation of iPSCs to cardiomyocytes (iPSC-CMs) has provided an intriguing option for the generation of patient-specific cardiac cells. In this review, a comprehensive overview of the currently published iPSC-CM models for hereditary heart disease is compiled and analysed. Besides the major findings of individual studies, detailed methodological information on iPSC generation, iPSC-CM differentiation, characterization, and maturation is included. Both, current advances in the field and challenges yet to overcome emphasize the potential of using patient-derived cell models to mimic genetic cardiac diseases.

Download Full-text

Cerebral organoids: ethical issues and consciousness assessment

Journal of Medical Ethics ◽

10.1136/medethics-2017-104555 ◽

2018 ◽

Vol 44 (9) ◽

pp. 606-610 ◽

Cited By ~ 26

Author(s):

Andrea Lavazza ◽

Marcello Massimini

Keyword(s):

Ethical Issues ◽

Personalised Medicine ◽

Three Dimensional ◽

Cell Types ◽

Human Embryos ◽

Specific Cell ◽

Neural Connections ◽

Structural And Functional Properties ◽

Organ Specific

Organoids are three-dimensional biological structures grown in vitro from different kinds of stem cells that self-organise mimicking real organs with organ-specific cell types. Recently, researchers have managed to produce human organoids which have structural and functional properties very similar to those of different organs, such as the retina, the intestines, the kidneys, the pancreas, the liver and the inner ear. Organoids are considered a great resource for biomedical research, as they allow for a detailed study of the development and pathologies of human cells; they also make it possible to test new molecules on human tissue. Furthermore, organoids have helped research take a step forward in the field of personalised medicine and transplants. However, some ethical issues have arisen concerning the origin of the cells that are used to produce organoids (ie, human embryos) and their properties. In particular, there are new, relevant and so-far overlooked ethical questions concerning cerebral organoids. Scientists have created so-called mini-brains as developed as a few-months-old fetus, albeit smaller and with many structural and functional differences. However, cerebral organoids exhibit neural connections and electrical activity, raising the question whether they are or (which is more likely) will one day be somewhat sentient. In principle, this can be measured with some techniques that are already available (the Perturbational Complexity Index, a metric that is directly inspired by the main postulate of the Integrated Information Theory of consciousness), which are used for brain-injured non-communicating patients. If brain organoids were to show a glimpse of sensibility, an ethical discussion on their use in clinical research and practice would be necessary.

Download Full-text

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

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666180410125850 ◽

2018 ◽

Vol 18 (4) ◽

pp. 246-255 ◽

Cited By ~ 1

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.

Download Full-text

Epigenetic reprogramming of cell identity: lessons from development for regenerative medicine

Clinical Epigenetics ◽

10.1186/s13148-021-01131-4 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Amitava Basu ◽

Vijay K. Tiwari

Keyword(s):

Regenerative Medicine ◽

Cell Types ◽

Direct Conversion ◽

Cellular Reprogramming ◽

Specific Cell ◽

Cell Type ◽

Pathological Conditions ◽

Gene Regulatory ◽

Induced Pluripotent ◽

And Function

AbstractEpigenetic mechanisms are known to define cell-type identity and function. Hence, reprogramming of one cell type into another essentially requires a rewiring of the underlying epigenome. Cellular reprogramming can convert somatic cells to induced pluripotent stem cells (iPSCs) that can be directed to differentiate to specific cell types. Trans-differentiation or direct reprogramming, on the other hand, involves the direct conversion of one cell type into another. In this review, we highlight how gene regulatory mechanisms identified to be critical for developmental processes were successfully used for cellular reprogramming of various cell types. We also discuss how the therapeutic use of the reprogrammed cells is beginning to revolutionize the field of regenerative medicine particularly in the repair and regeneration of damaged tissue and organs arising from pathological conditions or accidents. Lastly, we highlight some key challenges hindering the application of cellular reprogramming for therapeutic purposes.

Download Full-text

Hereditary Optic Neuropathies: Induced Pluripotent Stem Cell-Based 2D/3D Approaches

Genes ◽

10.3390/genes12010112 ◽

2021 ◽

Vol 12 (1) ◽

pp. 112

Author(s):

Marta García-López ◽

Joaquín Arenas ◽

M. Esther Gallardo

Keyword(s):

Stem Cell ◽

Retinal Ganglion Cells ◽

Pluripotent Stem Cell ◽

Ganglion Cells ◽

Genetic Disorders ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Underlying Mechanism ◽

Retinal Ganglion ◽

Induced Pluripotent

Inherited optic neuropathies share visual impairment due to the degeneration of retinal ganglion cells (RGCs) as the hallmark of the disease. This group of genetic disorders are caused by mutations in nuclear genes or in the mitochondrial DNA (mtDNA). An impaired mitochondrial function is the underlying mechanism of these diseases. Currently, optic neuropathies lack an effective treatment, and the implementation of induced pluripotent stem cell (iPSC) technology would entail a huge step forward. The generation of iPSC-derived RGCs would allow faithfully modeling these disorders, and these RGCs would represent an appealing platform for drug screening as well, paving the way for a proper therapy. Here, we review the ongoing two-dimensional (2D) and three-dimensional (3D) approaches based on iPSCs and their applications, taking into account the more innovative technologies, which include tissue engineering or microfluidics.

Download Full-text

Collagen gel three-dimensional matrices combined with adhesive proteins stimulate neuronal differentiation of mesenchymal stem cells

Journal of The Royal Society Interface ◽

10.1098/rsif.2010.0613 ◽

2011 ◽

Vol 8 (60) ◽

pp. 998-1010 ◽

Cited By ~ 38

Author(s):

Jae Ho Lee ◽

Hye-Sun Yu ◽

Gil-Su Lee ◽

Aeri Ji ◽

Jung Keun Hyun ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Neuronal Differentiation ◽

Large Population ◽

Three Dimensional ◽

Collagen Gel ◽

Cell Types ◽

Specific Cell ◽

Neuronal Outgrowth ◽

Adhesive Proteins

Three-dimensional gel matrices provide specialized microenvironments that mimic native tissues and enable stem cells to grow and differentiate into specific cell types. Here, we show that collagen three-dimensional gel matrices prepared in combination with adhesive proteins, such as fibronectin (FN) and laminin (LN), provide significant cues to the differentiation into neuronal lineage of mesenchymal stem cells (MSCs) derived from rat bone marrow. When cultured within either a three-dimensional collagen gel alone or one containing either FN or LN, and free of nerve growth factor (NGF), the MSCs showed the development of numerous neurite outgrowths. These were, however, not readily observed in two-dimensional culture without the use of NGF. Immunofluorescence staining, western blot and fluorescence-activated cell sorting analyses demonstrated that a large population of cells was positive for NeuN and glial fibrillary acidic protein, which are specific to neuronal cells, when cultured in the three-dimensional collagen gel. The dependence of the neuronal differentiation of MSCs on the adhesive proteins containing three-dimensional gel matrices is considered to be closely related to focal adhesion kinase (FAK) activation through integrin receptor binding, as revealed by an experiment showing no neuronal outgrowth in the FAK-knockdown cells and stimulation of integrin β1 gene. The results provided herein suggest the potential role of three-dimensional collagen-based gel matrices combined with adhesive proteins in the neuronal differentiation of MSCs, even without the use of chemical differentiation factors. Furthermore, these findings suggest that three-dimensional gel matrices might be useful as nerve-regenerative scaffolds.

Download Full-text

Developmental enhancers and chromosome topology

Science ◽

10.1126/science.aau0320 ◽

2018 ◽

Vol 361 (6409) ◽

pp. 1341-1345 ◽

Cited By ~ 139

Author(s):

Eileen E. M. Furlong ◽

Michael Levine

Keyword(s):

Three Dimensional ◽

Cell Types ◽

Living Cells ◽

Specific Cell ◽

Transcriptional Dynamics ◽

Spatiotemporal Expression ◽

Chromosome Topology ◽

Classical Models ◽

And Function ◽

Target Promoters

Developmental enhancers mediate on/off patterns of gene expression in specific cell types at particular stages during metazoan embryogenesis. They typically integrate multiple signals and regulatory determinants to achieve precise spatiotemporal expression. Such enhancers can map quite far—one megabase or more—from the genes they regulate. How remote enhancers relay regulatory information to their target promoters is one of the central mysteries of genome organization and function. A variety of contrasting mechanisms have been proposed over the years, including enhancer tracking, linking, looping, and mobilization to transcription factories. We argue that extreme versions of these mechanisms cannot account for the transcriptional dynamics and precision seen in living cells, tissues, and embryos. We describe emerging evidence for dynamic three-dimensional hubs that combine different elements of the classical models.

Download Full-text

Vertebrate retinal ganglion cells are selected from competent progenitors by the action of Notch

Development ◽

10.1242/dev.121.11.3637 ◽

1995 ◽

Vol 121 (11) ◽

pp. 3637-3650 ◽

Cited By ~ 33

Author(s):

C.P. Austin ◽

D.E. Feldman ◽

J.A. Ida ◽

C.L. Cepko

Keyword(s):

Cell Fate ◽

Ganglion Cells ◽

Notch Pathway ◽

Cell Types ◽

Projection Neurons ◽

Specific Cell ◽

Vitro Assay ◽

Notch Activity

The first cells generated during development of the vertebrate retina are the ganglion cells, the projection neurons of the retina. Although they are one of the most intensively studied cell types within the central nervous system, little is known of the mechanisms that determine ganglion cell fate. We demonstrate that ganglion cells are selected from a large group of competent progenitors that comprise the majority of the early embryonic retina and that differentiation within this group is regulated by Notch. Notch activity in vivo was diminished using antisense oligonucleotides or augmented using a retrovirally transduced constitutively active allele of Notch. The number of ganglion cells produced was inversely related to the level of Notch activity. In addition, the Notch ligand Delta inhibited retinal progenitors from differentiating as ganglion cells to the same degree as did activated Notch in an in vitro assay. These results suggest a conserved strategy for neurogenesis in the retina and describe a versatile in vitro and in vivo system with which to examine the action of the Notch pathway in a specific cell fate decision in a vertebrate.

Download Full-text

All About Cells

Bioprinting ◽

10.1093/oso/9780190943547.003.0003 ◽

2021 ◽

pp. 21-39

Author(s):

Kenneth Douglas

Keyword(s):

Stem Cells ◽

Cell Membrane ◽

Three Dimensional ◽

Scuba Diving ◽

Cell Types ◽

Two Dimensional ◽

Cell Organelles ◽

Outer Skin ◽

Induced Pluripotent ◽

Selective Access

Abstract: This chapter takes the reader on an imaginary scuba diving tour of the watery world of the cell and its surroundings, pointing out features such as the cytoskeleton (that forms the equivalent of the bones and muscles of our cells), the cell membrane (the outer skin of the cell), and the cell membrane’s embedded proteins that provide selective access to the interior of the cell—organelles (elfin versions our own organs). The chapter stresses the tumultuous action that occurs non-stop within the cells as proteins are assembled for use within and outside the cells. The chapter discusses stem cells, including the discovery of induced pluripotent stem cells. The chapter relates how cells differentiate to become dissimilar cell types, stresses the importance of three-dimensional study of cells (rather than two-dimensional study), and explains the different ways in which cells talk to each other.

Download Full-text

iPSC-Derived Liver Organoids: A Journey from Drug Screening, to Disease Modeling, Arriving to Regenerative Medicine

International Journal of Molecular Sciences ◽

10.3390/ijms21176215 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6215

Author(s):

Cristina Olgasi ◽

Alessia Cucci ◽

Antonia Follenzi

Keyword(s):

Regenerative Medicine ◽

Disease Modeling ◽

Three Dimensional ◽

Cell Types ◽

High Rate ◽

Limiting Factor ◽

Congenital Diseases ◽

Induced Pluripotent ◽

Liver Organoids ◽

Bioartificial Livers

Liver transplantation is the most common treatment for patients suffering from liver failure that is caused by congenital diseases, infectious agents, and environmental factors. Despite a high rate of patient survival following transplantation, organ availability remains the key limiting factor. As such, research has focused on the transplantation of different cell types that are capable of repopulating and restoring liver function. The best cellular mix capable of engrafting and proliferating over the long-term, as well as the optimal immunosuppression regimens, remain to be clearly well-defined. Hence, alternative strategies in the field of regenerative medicine have been explored. Since the discovery of induced pluripotent stem cells (iPSC) that have the potential of differentiating into a broad spectrum of cell types, many studies have reported the achievement of iPSCs differentiation into liver cells, such as hepatocytes, cholangiocytes, endothelial cells, and Kupffer cells. In parallel, an increasing interest in the study of self-assemble or matrix-guided three-dimensional (3D) organoids have paved the way for functional bioartificial livers. In this review, we will focus on the recent breakthroughs in the development of iPSCs-based liver organoids and the major drawbacks and challenges that need to be overcome for the development of future applications.

Download Full-text