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Pubmed Article
Three-dimensional neuroepithelial culture from human embryonic stem cells and its use for quantitative conversion to retinal pigment epithelium.
PLoS ONE
PUBLISHED: 01-24-2013
A goal in human embryonic stem cell (hESC) research is the faithful differentiation to given cell types such as neural lineages. During embryonic development, a basement membrane surrounds the neural plate that forms a tight, apico-basolaterally polarized epithelium before closing to form a neural tube with a single lumen. Here we show that the three-dimensional epithelial cyst culture of hESCs in Matrigel combined with neural induction results in a quantitative conversion into neuroepithelial cysts containing a single lumen. Cells attain a defined neuroepithelial identity by 5 days. The neuroepithelial cysts naturally generate retinal epithelium, in part due to IGF-1/insulin signaling. We demonstrate the utility of this epithelial culture approach by achieving a quantitative production of retinal pigment epithelial (RPE) cells from hESCs within 30 days. Direct transplantation of this RPE into a rat model of retinal degeneration without any selection or expansion of the cells results in the formation of a donor-derived RPE monolayer that rescues photoreceptor cells. The cyst method for neuroepithelial differentiation of pluripotent stem cells is not only of importance for RPE generation but will also be relevant to the production of other neuronal cell types and for reconstituting complex patterning events from three-dimensional neuroepithelia.
Authors: Erin M. Boisvert, Kyle Denton, Ling Lei, Xue-Jun Li.
Published: 04-14-2013
ABSTRACT
Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The differentiation process is initiated by breaking the human PSCs into clumps which round up to form aggregates when the cells are placed in a suspension culture. The aggregates are then grown in hESC medium from days 1-4 to allow for spontaneous differentiation. During this time, the cells have the capacity to become any of the three germ layers. From days 5-8, the cells are placed in a neural induction medium to push them into the neural lineage. Around day 8, the cells are allowed to attach onto 6 well plates and differentiate during which time the neuroepithelial cells form. These neuroepithelial cells can be isolated at day 17. The cells can then be kept as neurospheres until they are ready to be plated onto coverslips. Using a basic medium without any caudalizing factors, neuroepithelial cells are specified into telencephalic precursors, which can then be further differentiated into dorsal telencephalic progenitors and glutamatergic neurons efficiently. Overall, our system provides a tool to generate human glutamatergic neurons for researchers to study the development of these neurons and the diseases which affect them.
21 Related JoVE Articles!
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MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium
Authors: Whitney A. Greene, Alberto. Muñiz, Mark L. Plamper, Ramesh R. Kaini, Heuy-Ching Wang.
Institutions: JBSA Fort Sam Houston.
The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.
Molecular Biology, Issue 88, microRNA, microarray, human induced-pluripotent stem cells, retinal pigmented epithelium
51589
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Directed Dopaminergic Neuron Differentiation from Human Pluripotent Stem Cells
Authors: Pengbo Zhang, Ninuo Xia, Renee A. Reijo Pera.
Institutions: Stanford University School of Medicine, Stanford University School of Medicine.
Dopaminergic (DA) neurons in the substantia nigra pars compacta (also known as A9 DA neurons) are the specific cell type that is lost in Parkinson’s disease (PD). There is great interest in deriving A9 DA neurons from human pluripotent stem cells (hPSCs) for regenerative cell replacement therapy for PD. During neural development, A9 DA neurons originate from the floor plate (FP) precursors located at the ventral midline of the central nervous system. Here, we optimized the culture conditions for the stepwise differentiation of hPSCs to A9 DA neurons, which mimics embryonic DA neuron development. In our protocol, we first describe the efficient generation of FP precursor cells from hPSCs using a small molecule method, and then convert the FP cells to A9 DA neurons, which could be maintained in vitro for several months. This efficient, repeatable and controllable protocol works well in human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) from normal persons and PD patients, in which one could derive A9 DA neurons to perform in vitro disease modeling and drug screening and in vivo cell transplantation therapy for PD.
Neuroscience, Issue 91, dopaminergic neuron, substantia nigra pars compacta, midbrain, Parkinson’s disease, directed differentiation, human pluripotent stem cells, floor plate
51737
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In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors
Authors: Chiara Greggio, Filippo De Franceschi, Manuel Figueiredo-Larsen, Anne Grapin-Botton.
Institutions: Swiss Institute for Experimental Cancer Research, University of Copenhagen.
The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell´s tensegrity.
Developmental Biology, Issue 89, Pancreas, Progenitors, Branching Epithelium, Development, Organ Culture, 3D Culture, Diabetes, Differentiation, Morphogenesis, Cell organization, Beta Cell.
51725
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Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells
Authors: Nadja Zeltner, Fabien G. Lafaille, Faranak Fattahi, Lorenz Studer.
Institutions: Sloan-Kettering Institute for Cancer Research, The Rockefeller University.
Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.
Neuroscience, Issue 87, Embryonic Stem Cells (ESCs), Pluripotent Stem Cells, Induced Pluripotent Stem Cells (iPSCs), Neural Crest, Peripheral Nervous System (PNS), pluripotent stem cells, neural crest cells, in vitro differentiation, disease modeling, differentiation protocol, human embryonic stem cells, human pluripotent stem cells
51609
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High Efficiency Differentiation of Human Pluripotent Stem Cells to Cardiomyocytes and Characterization by Flow Cytometry
Authors: Subarna Bhattacharya, Paul W. Burridge, Erin M. Kropp, Sandra L. Chuppa, Wai-Meng Kwok, Joseph C. Wu, Kenneth R. Boheler, Rebekah L. Gundry.
Institutions: Medical College of Wisconsin, Stanford University School of Medicine, Medical College of Wisconsin, Hong Kong University, Johns Hopkins University School of Medicine, Medical College of Wisconsin.
There is an urgent need to develop approaches for repairing the damaged heart, discovering new therapeutic drugs that do not have toxic effects on the heart, and improving strategies to accurately model heart disease. The potential of exploiting human induced pluripotent stem cell (hiPSC) technology to generate cardiac muscle “in a dish” for these applications continues to generate high enthusiasm. In recent years, the ability to efficiently generate cardiomyogenic cells from human pluripotent stem cells (hPSCs) has greatly improved, offering us new opportunities to model very early stages of human cardiac development not otherwise accessible. In contrast to many previous methods, the cardiomyocyte differentiation protocol described here does not require cell aggregation or the addition of Activin A or BMP4 and robustly generates cultures of cells that are highly positive for cardiac troponin I and T (TNNI3, TNNT2), iroquois-class homeodomain protein IRX-4 (IRX4), myosin regulatory light chain 2, ventricular/cardiac muscle isoform (MLC2v) and myosin regulatory light chain 2, atrial isoform (MLC2a) by day 10 across all human embryonic stem cell (hESC) and hiPSC lines tested to date. Cells can be passaged and maintained for more than 90 days in culture. The strategy is technically simple to implement and cost-effective. Characterization of cardiomyocytes derived from pluripotent cells often includes the analysis of reference markers, both at the mRNA and protein level. For protein analysis, flow cytometry is a powerful analytical tool for assessing quality of cells in culture and determining subpopulation homogeneity. However, technical variation in sample preparation can significantly affect quality of flow cytometry data. Thus, standardization of staining protocols should facilitate comparisons among various differentiation strategies. Accordingly, optimized staining protocols for the analysis of IRX4, MLC2v, MLC2a, TNNI3, and TNNT2 by flow cytometry are described.
Cellular Biology, Issue 91, human induced pluripotent stem cell, flow cytometry, directed differentiation, cardiomyocyte, IRX4, TNNI3, TNNT2, MCL2v, MLC2a
52010
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Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis
Authors: Kevin G. Chen, Rebecca S. Hamilton, Pamela G. Robey, Barbara S. Mallon.
Institutions: National Institutes of Health, National Institutes of Health.
Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.
Stem Cell Biology, Issue 89, Pluripotent stem cells, human embryonic stem cells, induced pluripotent stem cells, cell culture, non-colony type monolayer, single cell, plating efficiency, Rho-associated kinase, Y-27632, transfection, transduction
51519
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Generation of Myospheres From hESCs by Epigenetic Reprogramming
Authors: Sonia Albini, Pier Lorenzo Puri.
Institutions: Sanford-Burnham Institute for Medical Research, IRCCS Fondazione Santa Lucia.
Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based therapies and for a "disease in a dish" model of human neuromuscular diseases. Major hurdles, such as low abundance and heterogeneity of the population of interest, as well as a lack of protocols for the formation of three-dimensional contractile structures, have limited the applications of stem cells for neuromuscular disorders. We have designed a protocol that overcomes these limits by ectopic introduction of defined factors in hESCs - the muscle determination factor MyoD and SWI/SNF chromatin remodeling complex component BAF60C - that are able to reprogram hESCs into skeletal muscle cells. Here we describe the protocol established to generate hESC-derived myoblasts and promote their clustering into tridimensional miniaturized structures (myospheres) that functionally mimic miniaturized skeletal muscles7.
Bioengineering, Issue 88, Tissues, Cells, Embryonic Structures, Musculoskeletal System, Musculoskeletal Diseases, hESC, epinegetics, Skeletal Myogenesis, Myosphere, Chromatin, Lentivirus, Infection
51243
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A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue
Authors: Brandon C. Shelley, Geneviève Gowing, Clive N. Svendsen.
Institutions: Cedars-Sinai Medical Center.
A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as “chopping” that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.
Neuroscience, Issue 88, neural progenitor cell, neural precursor cell, neural stem cell, passaging, neurosphere, chopping, stem cell, neuroscience, suspension culture, good manufacturing practice, GMP
51219
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Enrichment and Purging of Human Embryonic Stem Cells by Detection of Cell Surface Antigens Using the Monoclonal Antibodies TG30 and GCTM-2
Authors: Juan Carlos Polanco, Bei Wang, Qi Zhou, Hun Chy, Carmel O'Brien, Andrew L. Laslett.
Institutions: CSIRO.
Human embryonic stem cells (hESC) can self-renew indefinitely in vitro, and with the appropriate cues can be induced to differentiate into potentially all somatic cell lineages. Differentiated hESC derivatives can potentially be used in transplantation therapies to treat a variety of cell-degenerative diseases. However, hESC differentiation protocols usually yield a mixture of differentiated target and off-target cell types as well as residual undifferentiated cells. For the translation of differentiated hESC-derivatives from the laboratory to the clinic, it is important to be able to discriminate between undifferentiated (pluripotent) and differentiated cells, and generate methods to separate these populations. Safe application of hESC-derived somatic cell types can only be accomplished with pluripotent stem cell-free populations, as residual hESCs could induce tumors known as teratomas following transplantation. Towards this end, here we describe a methodology to detect pluripotency associated cell surface antigens with the monoclonal antibodies TG30 (CD9) and GCTM-2 via fluorescence activated cell sorting (FACS) for the identification of pluripotent TG30Hi-GCTM-2Hi hESCs using positive selection. Using negative selection with our TG30/GCTM-2 FACS methodology, we were able to detect and purge undifferentiated hESCs in populations undergoing very early-stage differentiation (TG30Neg-GCTM-2Neg). In a further study, pluripotent stem cell-free samples of differentiated TG30Neg-GCTM-2Neg cells selected using our TG30/GCTM-2 FACS protocol did not form teratomas once transplanted into immune-compromised mice, supporting the robustness of our protocol. On the other hand, TG30/GCTM-2 FACS-mediated consecutive passaging of enriched pluripotent TG30Hi-GCTM-2Hi hESCs did not affect their ability to self-renew in vitro or their intrinsic pluripotency. Therefore, the characteristics of our TG30/GCTM-2 FACS methodology provide a sensitive assay to obtain highly enriched populations of hPSC as inputs for differentiation assays and to rid potentially tumorigenic (or residual) hESC from derivative cell populations.
Stem Cell Biology, Issue 82, Stem cells, cell surface antigens, antibodies, FACS, purging stem cells, differentiation, pluripotency, teratoma, human embryonic stem cells (hESC)
50856
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Derivation and Characterization of a Transgene-free Human Induced Pluripotent Stem Cell Line and Conversion into Defined Clinical-grade Conditions
Authors: Jason P. Awe, Agustin Vega-Crespo, James A. Byrne.
Institutions: University of California, Los Angeles (UCLA), University of California, Los Angeles (UCLA).
Human induced pluripotent stem cells (hiPSCs) can be generated with lentiviral-based reprogramming methodologies. However, traces of potentially oncogenic genes remaining in actively transcribed regions of the genome, limit their potential for use in human therapeutic applications1. Additionally, non-human antigens derived from stem cell reprogramming or differentiation into therapeutically relevant derivatives preclude these hiPSCs from being used in a human clinical context2. In this video, we present a procedure for reprogramming and analyzing factor-free hiPSCs free of exogenous transgenes. These hiPSCs then can be analyzed for gene expression abnormalities in the specific intron containing the lentivirus. This analysis may be conducted using sensitive quantitative polymerase chain reaction (PCR), which has an advantage over less sensitive techniques previously used to detect gene expression differences3. Full conversion into clinical-grade good manufacturing practice (GMP) conditions, allows human clinical relevance. Our protocol offers another methodology—provided that current safe-harbor criteria will expand and include factor-free characterized hiPSC-based derivatives for human therapeutic applications—for deriving GMP-grade hiPSCs, which should eliminate any immunogenicity risk due to non-human antigens. This protocol is broadly applicable to lentiviral reprogrammed cells of any type and provides a reproducible method for converting reprogrammed cells into GMP-grade conditions.
Stem Cell Biology, Issue 93, Human induced pluripotent stem cells, STEMCCA, factor-free, GMP, xeno-free, quantitative PCR
52158
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Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye
Authors: Katherine J. Wert, Jessica M. Skeie, Richard J. Davis, Stephen H. Tsang, Vinit B. Mahajan.
Institutions: Columbia University , Columbia University , University of Iowa , University of Iowa .
The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.
Stem Cell Biology, Issue 69, Medicine, Ophthalmology, Anatomy, Physiology, Cellular Biology, Genetics, mouse, subretinal injection, iPS cells, stem cells, retina, eye, gene therapy
4286
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In vivo-like Organotypic Murine Retinal Wholemount Culture
Authors: Sebastian Gustmann, Nicole Dünker.
Institutions: University of Duisburg-Essen.
Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages. Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used. By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5. In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.
Neuroscience, Issue 35, eye, retina, organotypic culture, wholemount, dissociation, cytospin
1634
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Microdissection of Zebrafish Embryonic Eye Tissues
Authors: Liyun Zhang, Yuk Fai Leung.
Institutions: Purdue University.
Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 μm, while the diameter of the lens is less 100 μm. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.
Developmental biology, Issue 40, zebrafish, retina, retinal pigment epithelium, microdissection, development, gene expression, microarrays
2028
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Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology
Authors: Arvydas Maminishkis, Sheldon S. Miller.
Institutions: National Institutes of Health.
We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.
Neuroscience, Issue 45, epithelia, cultures, fluid transport, channels, polarization, edema, retina, detachment, monolayer
2032
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Isolation of Retinal Stem Cells from the Mouse Eye
Authors: Brenda L.K. Coles, Derek van der Kooy.
Institutions: University of Toronto.
The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment4; however the cells are still capable of forming the different cell types found within the neural retina1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury5, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.
Cellular Biology, Issue 43, Stem Cells, Eye, Ciliary Epithelium, Tissue Culture, Mouse
2209
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Dissection of a Mouse Eye for a Whole Mount of the Retinal Pigment Epithelium
Authors: Alison Claybon, Alexander J. R. Bishop.
Institutions: Greehey Children's Cancer Research Institute and Department of Cellular and Structural Biology.
The retinal pigment epithelium (RPE) lies at the back of the mammalian eye, just under the neural retina, which contains the photoreceptors (rods and cones). The RPE is a monolayer of pigmented cuboidal cells and associates closely with the neural retina just above it. This association makes the RPE of great interest to researchers studying retinal diseases. The RPE is also the site of an in vivo assay of homology-directed DNA repair, the pun assay. The mouse eye is particularly difficult to dissect due to its small size (about 3.5mm in diameter) and its spherical shape. This article demonstrates in detail a procedure for dissection of the eye resulting in a whole mount of the RPE. In this procedure, we show how to work with, rather than against, the spherical structure of the eye. Briefly, the connective tissue, muscle, and optic nerve are removed from the back of the eye. Then, the cornea and lens are removed. Next, strategic cuts are made that result in significant flattening of the remaining tissue. Finally, the neural retina is gently lifted off, revealing an intact RPE, which is still attached to the underlying choroid and sclera. This whole mount can be used to perform the pun assay or for immunohistochemistry or immunofluorescent assessment of the RPE tissue.
Neuroscience, Issue 48, mouse, dissection, eye, retinal pigment epithelium, flat mount, whole mount, RPE
2563
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Organotypic Culture of Full-thickness Adult Porcine Retina
Authors: Jianfeng Wang, Anton M. Kolomeyer, Marco A. Zarbin, Ellen Townes-Anderson.
Institutions: University of Medicine and Dentistry of New Jersey - UMDNJ, University of Medicine and Dentistry of New Jersey - UMDNJ.
There is a recognized demand for in vitro models that can replace or reduce animal experiments. Porcine retina has a similar neuronal structure to human retina and is therefore a valuable species for studying mechanisms of human retinal injury and degenerative disease. Here we describe a cost-effective technique for organotypic culture of adult porcine retina isolated from eyes obtained from an abattoir. After removing the anterior segment, a trephine blade was used to create multiple neural retina-Bruch's membrane-RPE-choroid-sclera explants from the posterior segment of adult porcine eyes. A piece of sterile filter paper was used to lift the neural retina off from each explant. The filter paper-retina complex was cultured (photoreceptor side up) atop an insert, which was held away from the bottom of the culture dish by a custom-made stand. The stand allows for good circulation of the culture medium to both sides of the retina. Overall, this procedure is simple, reproducible, and permits preservation of native retinal structure for at least seven days, making it a useful model for a variety of morphological, pharmacological, and biochemical studies on mammalian retina.
Neuroscience, Issue 49, Retina, in vitro, Porcine, Photoreceptor
2655
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Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction
Authors: Xuejun H. Parsons, Yang D. Teng, James F. Parsons, Evan Y. Snyder, David B. Smotrich, Dennis A. Moore.
Institutions: San Diego Regenerative Medicine Institute, Xcelthera, Harvard Medical School, Division of SCI Research, VA Boston Healthcare System, Sanford-Burnham Medical Research Institute, La Jolla IVF.
There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system (CNS) structure and circuitry in today's healthcare industry. Cell-based therapies hold great promise to restore the lost nerve tissue and function for CNS disorders. However, cell therapies based on CNS-derived neural stem cells have encountered supply restriction and difficulty to use in the clinical setting due to their limited expansion ability in culture and failing plasticity after extensive passaging1-3. Despite some beneficial outcomes, the CNS-derived human neural stem cells (hNSCs) appear to exert their therapeutic effects primarily by their non-neuronal progenies through producing trophic and neuroprotective molecules to rescue the endogenous cells1-3. Alternatively, pluripotent human embryonic stem cells (hESCs) proffer cures for a wide range of neurological disorders by supplying the diversity of human neuronal cell types in the developing CNS for regeneration1,4-7. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity7-10. In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic11-13. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules14 (please see a schematic in Fig. 1). Retinoic acid (RA) does not induce neuronal differentiation of undifferentiated hESCs maintained on feeders1, 14. And unlike mouse ESCs, treating hESC-differentiated embryoid bodies (EBs) only slightly increases the low yield of neurons1, 14, 15. However, after screening a variety of small molecules and growth factors, we found that such defined conditions rendered retinoic acid (RA) sufficient to induce the specification of neuroectoderm direct from pluripotent hESCs that further progressed to neuroblasts that generated human neuronal progenitors and neurons in the developing CNS with high efficiency (Fig. 2). We defined conditions for induction of neuroblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human neuronal cells across the spectrum of developmental stages for cell-based therapeutics.
Neuroscience, Issue 56, stem cell, human embryonic stem cell, human, neuronal progenitor, neuron, human pluripotent cell, neuronal differentiation, small molecule induction, cell culture, cell therapy
3273
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An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium
Authors: Jessica T. Chang, Hazel Sive.
Institutions: Massachusetts Institute of Technology, Whitehead Institute of Biomedical Research.
The brain ventricular system is conserved among vertebrates and is composed of a series of interconnected cavities called brain ventricles, which form during the earliest stages of brain development and are maintained throughout the animal's life. The brain ventricular system is found in vertebrates, and the ventricles develop after neural tube formation, when the central lumen fills with cerebrospinal fluid (CSF) 1,2. CSF is a protein rich fluid that is essential for normal brain development and function3-6. In zebrafish, brain ventricle inflation begins at approximately 18 hr post fertilization (hpf), after the neural tube is closed. Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8, while claudin 5a is necessary for tight junction formation 9. Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation. To investigate the regulation of permeability during zebrafish brain ventricle inflation, we developed a ventricular dye retention assay. This method uses brain ventricle injection in a living zebrafish embryo, a technique previously developed in our lab10, to fluorescently label the cerebrospinal fluid. Embryos are then imaged over time as the fluorescent dye moves through the brain ventricles and neuroepithelium. The distance the dye front moves away from the basal (non-luminal) side of the neuroepithelium over time is quantified and is a measure of neuroepithelial permeability (Figure 1). We observe that dyes 70 kDa and smaller will move through the neuroepithelium and can be detected outside the embryonic zebrafish brain at 24 hpf (Figure 2). This dye retention assay can be used to analyze neuroepithelial permeability in a variety of different genetic backgrounds, at different times during development, and after environmental perturbations. It may also be useful in examining pathological accumulation of CSF. Overall, this technique allows investigators to analyze the role and regulation of permeability during development and disease.
Neuroscience, Issue 68, Zebrafish, neuroepithelium, brain ventricle, permeability
4242
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Propagation of Human Embryonic Stem (ES) Cells
Authors: Laurence Daheron.
Institutions: MGH - Massachusetts General Hospital.
Cellular Biology, Issue 1, ES, embryonic stem cells, tissue culture
119
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ES Cell-derived Neuroepithelial Cell Cultures
Authors: Shreeya Karki, Jan Pruszak, Ole Isacson, Kai C Sonntag.
Institutions: Harvard Medical School.
ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).
Cellular Biology, issue 1, embryonic stem (ES) cells, rosettes, neuroepithelial precursors, stromal cells, differentiation
118
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