<h2 class="jove_title"><a href="https://www.jove.com/video/53174">Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes</a></h2>Sandy S.C. Hung, Alice P&#233;bay, Raymond C.B. WongCentre for Eye Research Australia &amp; Department of Ophthalmology, University of MelbourneThis manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors. <h2 class="jove_title"><a href="https://www.jove.com/video/53271">Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development</a></h2>Joseph S. Uzarski1,2, Jimmy Su1,2,3,4, Yan Xie1,2, Zheng Jenny Zhang1,2, Heather H. Ward5, Angela Wandinger-Ness6, William M. Miller7,8, Jason A. Wertheim1,2,3,4,8,91Comprehensive Transplant Center, Feinberg School of Medicine, Northwestern University, 2Department of Surgery, Feinberg School of Medicine, Northwestern University, 3Department of Biomedical Engineering, Northwestern University, 4Simpson Querrey Institute for BioNanotechnology in Medicine, Northwestern University, 5Department of Internal Medicine, University of New Mexico HSC, 6Department of Pathology, University of New Mexico HSC, 7Department of Chemical and Biological Engineering, Northwestern University, 8Chemistry of Life Processes Institute, Northwestern University, 9Department of Surgery, Jesse Brown VA Medical CenterThis protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.<h2 class="jove_title"><a href="https://www.jove.com/video/52898">A Treatment Package without Escape Extinction To Address Food Selectivity</a></h2>Jessica Weber1, Anibal Gutierrez, Jr.21Florida International University, 2Department of Psychology and the Center for Children and Families, Florida International UniversityFeeding difficulties are a common problem for children with developmental disorders, including autism, and behavioral interventions often include escape extinction. Recent research has begun to evaluate treatments that do not include escape extinction. This manuscript describes a multicomponent treatment package that does not use escape extinction to treat feeding difficulties.<h2 class="jove_title"><a href="https://www.jove.com/video/53204">A Novel Culture Model for Human Pluripotent Stem Cell Propagation on Gelatin in Placenta-conditioned Media</a></h2>Ji-Hye Jung, Byung Soo KimDepartment of Biomedical Science, Graduate School of Medicine, Korea UniversityThis protocol provides a simple and efficient way to propagate human pluripotent stem cells (hPSCs) using only conditioned media derived from the human placenta in a gelatin-coated dish without additional exogenous supplementation or hPSC-specific synthetic substrata.

august 2015 - this month in jove: isolating stem cells, bioengineering the kidney, and getting kids to eat carrots

Here's a look at what's coming up in the <a href="http://www.jove.com/archive/102">August 2015 issue</a> of <a href="http://www.jove.com/">JoVE: The Journal of Visualized Experiments</a>.
In <a href="http://www.jove.com/devbio">JoVE Developmental Biology</a>, a major goal in stem cell research is to grow human pluripotent stem cells (hPSCs) in fully humanized conditions without using feeder cells, exogenous supplements, or synthetic substrates. This will eliminate the risk of contamination by animal cells and facilitate economical mass production for therapeutic applications. <a href="http://www.jove.com/video/53204">Jung and Kim</a> previously showed that chorionic cells from the human placenta could support the propagation of hPSCs. So this month, they isolate and culture placental chorionic cells and use the placenta-conditioned media to culture the hPSCs. The result is an almost completely humanized culture system that can advance stem cell research and regenerative medicine.
Also in <a href="http://www.jove.com/devbio">JoVE Developmental Biology</a>, the ability to genetically reprogram differentiated cells into stem-like states has revolutionized the field of regenerative medicine. Induced pluripotent stem cells (iPSCs) can be derived from many different cell types, including lymphocytes from whole blood and keratinocytes from skin samples. But these require pretty invasive extraction methods, so <a href="http://www.jove.com/video/53174">Hung et al.</a> focus on the keratinocytes from hair follicles, which can be harvested simply by plucking a few hairs. Our authors culture the follicles to isolate keratinocytes, and transfect them with non-viral episomal vectors to produce iPSCs. These could be used for disease modeling and for deriving patient-specific stem cells.
<a href="http://www.jove.com/bioengineering">JoVE Bioengineering</a> also applies to regenerative medicine with a protocol by <a href="http://www.jove.com/video/53271">Uzarski et al.</a> for bioengineering kidney tissues. This involves carefully removing the cells from kidneys by perfusion. The decellularized tissue retains much of the kidney's intricate structure, so it serves a biological scaffold for new renal cells. The kidney scaffolds are recellularized with human renal cells, which repopulate the matrix, proliferate, and survive in perfusion culture for over a week. With a shortage of donor kidneys available for transplantation, patients with renal failure may benefit from kidney grafts made from their own cells.
In <a href="http://www.jove.com/behavior">JoVE Behavior</a>, feeding disorders are common in young children, especially those with developmental delays. So <a href="http://www.jove.com/video/52898">Weber and Gutierrez</a> demonstrate a treatment protocol that includes three different procedures for presenting foods: One procedure involves several successive steps that approximate eating. In another, preferred foods are always placed on the left side. This is designed to foster compliance with foods served on the left. In the third procedure, the non-preferred food is placed on top of the preferred one, and always served from the left. Parents, teachers, and clinicians can try this simple protocol before seeking more specialized treatment.
You've just had a sneak peek of the <a href="http://www.jove.com/archive/102">August 2015 issue</a> of <a href="http://www.jove.com/">JoVE</a>. Visit the website to see the full-length articles, plus many more, in <a href="http://www.jove.com/">JoVE: The Journal of Visualized Experiments</a>.

Watch this Scientific Journal Video about August 2015 - This Month in JoVE: Isolating Stem Cells, Bioengineering the Kidney, and Getting Kids to eat Carrots at JoVE.com

August 2015 - This Month in JoVE: Isolating Stem Cells, Bioengineering the Kidney, and Getting Kids to eat Carrots

Here's a look at what's coming up in the August 2015 issue of JoVE: The Journal of Visualized Experiments.
In JoVE Developmental Biology, a major goal in ...

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2.0K ビュー.  Massachusetts Eye and Ear. August 2015 - This Month in JoVE: Isolating Stem Cells, Bioengineering the Kidney, and Getting Kids to eat Carrots

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isolating stem cells from soft musculoskeletal tissues

Isolating Stem Cells from Soft Musculoskeletal Tissues

Isolating adult stem cells from musculoskeletal soft tissues based on the cell's adherence speed to...

generating kidney organoids in suspension from induced pluripotent stem cells

Author Spotlight: Optimizing iPSC Differentiation for Efficient Production to Generate Kidney Organoids 

This protocol presents a comprehensive and efficient method for producing kidney organoids from induced pluripotent stem cells (iPSCs) using suspension culture conditions. The primary emphasis of this study lies in the determination of the initial cell density and the WNT agonist concentration, thereby benefiting investigators interested in kidney organoid...

Generating Kidney Organoids in Suspension from Induced Pluripotent Stem Cells

isolating, sequencing and analyzing extracellular micrornas from human mesenchymal stem cells

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like...

isolating intestinal stem cells from adult drosophila midguts by facs to study stem cell behavior during aging

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

Understanding the endogenous molecular changes in adult stem cells during aging requires isolating the cells of interest. The method described here presents a simple and robust approach to enrich for and isolate Drosophila intestinal stem cells and the enteroblast progenitor cells by FACS at any time point during aging.

Isolating Intestinal Stem Cells from Adult Drosophila Midguts by FACS to Study Stem Cell Behavior During Aging

isolating hair follicle stem cells and epidermal keratinocytes from dorsal mouse skin

Isolating Hair Follicle Stem Cells and Epidermal Keratinocytes from Dorsal Mouse Skin

An ideal model for studying adult stem cell biology is the mouse hair follicle. Here we present a protocol for isolating different populations of hair follicles stem cells and epidermal keratinocytes, employing enzymatic digestion of mouse dorsal skin followed by FACS...

isolating cord blood hematopoietic stem cells to generate human immune system mice

This video demonstrates a technique for generating human immune system (HIS) mice. CD34+ hematopoietic stem cells (HSCs) are isolated from human cord blood buffy coat cells using antibody-coated magnetic beads. The CD34+ HSCs are expanded in a growth medium enriched with cytokines and cryopreserved. Finally, the thawed HSCs are injected into an immunodeficient mouse pup to establish the HIS mouse...

Isolating Cord Blood Hematopoietic Stem Cells to Generate Human Immune System Mice

deriving human kidney podocytes from induced pluripotent stem cells: a procedure for directed differentiation of mature kidney podocytes from stem cells

In this video, we describe the protocol to derive human kidney podocytes from induced pluripotent stem cells through cell differentiation under defined chemical conditions. The generated podocytes can be used for nephrotoxicity testing and disease...

Deriving Human Kidney Podocytes from Induced Pluripotent Stem Cells: A Procedure for Directed Differentiation of Mature Kidney Podocytes from Stem Cells

isolating nasal olfactory stem cells from rodents or humans

Isolating Nasal Olfactory Stem Cells from Rodents or Humans

We describe here a method for biopsying olfactory mucosa from rat and human nasal cavities. These biopsies can be used for either identifying molecular anomalies in brain diseases or isolating multipotent adult stem cells that can be utilized for cell transplantation in animal models of brain...

isogenic kidney glomerulus chip engineered from human induced pluripotent stem cells

Isogenic Kidney Glomerulus Chip Engineered from Human Induced Pluripotent Stem Cells

Presented here is a protocol to engineer a personalized organ-on-a-chip system that recapitulates the structure and function of the kidney glomerular filtration barrier by integrating genetically matched epithelial and vascular endothelial cells differentiated from human induced pluripotent stem cells. This bioengineered system can advance kidney precision medicine and related...

a simplified method for generating kidney organoids from human pluripotent stem cells

A Simplified Method for Generating Kidney Organoids from Human Pluripotent Stem Cells

Here we describe a protocol to generate kidney organoids from human pluripotent stem cells (hPSCs). This protocol generates kidney organoids within two weeks. The resulting kidney organoids can be cultured in large-scale spinner flasks or multi-well magnetic stir plates for parallel drug-testing...

isolating and analyzing cells of the pancreas mesenchyme by flow cytometry

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry

Here, we describe a method for the isolation of cells in the pancreas microenvironment from embryonic, neonatal and adult mouse tissue, focusing on the isolation of mesenchymal cells. This method allows profiling of cell gene expression and protein secretion in order to elucidate the extrinsic signals that regulate pancreas development, function, and...

Next, add 5 milliliters of magnetic cell separator buffer per 1 x 108 cells and spin at 360 g for 10 minutes at 4 degrees Celsius. Repeat the wash step and resuspend the pellet in 500 microliters per 100 million cells of magnetic cell separator buffer in a 15-milliliter conical tube labeled &#39;unfractionated.&#39; Label two more 15-milliliter tubes&#8212;CD34-negative and CD34-positive. Place the three tubes on a cooling...

Add 1 milliliter of cells to each well of the basement membrane matrix 2-coated 12-well plates and gently shake the plates to distribute the cells more evenly. Then, place the plate into the cell culture incubator. On days 2 to 15 of the differentiation, replace the mesoderm induction medium with 1 milliliter of intermediate mesoderm induction medium per...

isolating adipose-derived stem cells from murine periaortic adipose tissue

This video demonstrates a technique for isolating and culturing fluorescently labeled neural crest-derived adipose-derived stem cells (ADSCs) from murine periaortic arch adipose tissue. Following isolation, the NCADSCs are detected, sorted using fluorescence-activated cell sorting, and subsequently cultured on a plate, promoting cell...

Isolating Adipose-Derived Stem Cells from Murine Periaortic Adipose Tissue

isolation of double negative &#945;&#946; t cells from the kidney

Isolation of Double Negative &amp;#945;&amp;#946; T Cells from the Kidney

DNabT cells are rare among peripheral T cells; however, they are abundant in certain non-lymphoid tissues. Difficulty of isolating DN T cells from non-lymphoid tissue hinders their functional analysis despite increasing recognized pathophysiologic significance. We describe a novel protocol for isolation of highly purified DN T cells from murine...

Isolation of Double Negative &#945;&#946; T Cells from the Kidney

selecting and isolating colonies of human induced pluripotent stem cells reprogrammed from adult fibroblasts

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81...

reliable and high efficiency extraction of kidney immune cells

Reliable and High Efficiency Extraction of Kidney Immune Cells

Techniques that are reliable and efficient for the isolation of kidney immune cells are needed for downstream applications. This requires surface antibody labeling of a small number of kidney immune cells. Herein, we describe a concise method for isolation of kidney immune cells that seemingly achieves this...

normothermic ex vivo kidney perfusion for the preservation of kidney grafts prior to transplantation

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

The severe organ shortage has resulted in increased use of marginal kidney grafts for transplantation. This has triggered interest in alternative storage methods, since marginal grafts especially tolerate cold storage poorly. The technique of normothermic ex vivo kidney perfusion (NEVKP) represents a novel preservation method for kidney grafts prior to...

Normothermic Ex Vivo Kidney Perfusion for the Preservation of Kidney Grafts prior to Transplantation

bioengineering human microvascular networks in immunodeficient mice

Bioengineering Human Microvascular Networks in Immunodeficient Mice

Here, we describe a methodology to deliver human cord blood-derived endothelial colony-forming cells (ECFCs) and bone marrow-derived mesenchymal stem cells (MSCs), embedded in a collagen/fibronectin gel, subcutaneously into immunodeficient mice. This cell/gel combination generates a human vascular network that connects with the mouse...

guided differentiation of mature kidney podocytes from human induced pluripotent stem cells under chemically defined conditions

Guided Differentiation of Mature Kidney Podocytes from Human Induced Pluripotent Stem Cells Under Chemically Defined Conditions

Presented here is a chemically defined protocol for the derivation of human kidney podocytes from induced pluripotent stem cells with high efficiency (&#62;90%) and independent of genetic manipulations or subpopulation selection. This protocol produces the desired cell type within 26 days and could be useful for nephrotoxicity testing and disease...

August 2012: This Month in JoVE

Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and mirrors to refract and direct light to optical sensors. The August 2012 issue of JoVE (Journal of Visualized Experiments) is marked by the third publication from the Ozcan Lab (University of California, Los Angeles) on their lens free lens-free "on-chip" microscopy platform, which they have pioneered.
Back in 2009, the Ozcan lab published their first article, which demonstrated the use of a lens-free imaging platform that could image cells within whole blood. This imaging technique involves placing a sample directly on an optoelectric sensor array, like the CCD or CMOS chips found in the standard camcorder. Then, LED illumination from above the sample casts shadows on the chip, which are recorded as digital holograms that can be subjected to image processing. Not only does this lensless imaging system allow for an extremely large field of view (FOV of 0.6-8 cm2), the lack of bulky optical components makes it excellent for microfluidic applications. The Ozcan group demonstrates this in their follow-up 2011 JoVE article, in which fluorescently labeled white blood cells are imaged within a microfluidic chip.
This August in JoVE, the Ozcan group demonstrates further advancements to their lens-free microscopy platform by applying it to tomographic imaging of the nematode, C. elegans. By changing the angle of illumination, using a motorized rotation stage, the Ozcan group shows how multiple holographic slices may be generated for a sample-which, following processing, can be combined into tomograms. The Ozcan group confirms that their 3D reconstruction of the worm is successful by citing that the pharyngeal tube, a 5-micron-thick structure, is only visible in a single slice and in a 3D reconstructed cross section.
The compact and inexpensive nature of the lens-free imaging systems - designed and assembled by the Ozcan group - make them well suited for point-of-care diagnostic applications, or even cell phone based microscopy that can be performed in the field.
At the Ernest Gallo Clinic and Research Center at University of California, San Francisco JoVE learns how to visualize morphological changes in single cells using three-dimensional fluorescence microscopy and computer analysis. In this procedure, cells are given agonist, antagonist plus agonist, or no treatment, and prepared for immunofluorescence microscopy. Three-dimensional images are acquired with the help of a two-photon laser system, and the fluorescence data are processed on the computer using Imaris and Matlab.
To demonstrate the power of this technique, our authors quantify single-cell phenotypic changes in transfected HEK293 cells. Three-dimensional morphometric analysis shows dramatic changes in cells treated with agonist. These changes are not always apparent when using conventional 2D analysis. This technique allows morphological changes in single cells to be quantified and characterized in response to pharmacologic stimuli, offering a powerful new assessment tool to the arsenal of drug discovery research.
This month, in JoVE Applied Physics, we travel to South Carolina to visit the School of Materials Science and Engineering at Clemson University. Here, researchers show us how to construct lithium batteries for laboratory research.
The basic components of a lithium coin cell are the working electrode, the lithium electrolyte, and a lithium foil counter electrode. The different components of the coin cell are carefully assembled and sealed with a crimping machine. The finished coin cell can then be characterized in a battery tester. Using their homemade coin cells, our researchers can experiment with using different materials to make the cathodes of lithium coin cells.
This month in JoVE Immunology and Infection, we head to Basel, Switzerland to witness two techniques for diagnosing helminth infections. We also see how investigators perform the shuttle run test in children, in order to assess the effects of these infections on physical fitness.
Helminths are parasitic worms that can live inside humans and animals. Many species infect the intestinal tract, and may be transmitted through soil. Our authors demonstrate the Kato-Katz technique, which is used for diagnosing several common soil-transmitted helminths. They also demonstrate the Baermann technique, which uses light to stimulate the migration of certain types of phototactic helminths out of sample preparations. The various types of helminth species can subsequently be determined based on morphology.
Our authors then demonstrate how to set up and perform a 20-meter shuttle run test for children, and to measure the impact of helminth infections on physical fitness. This method can help determine the true burden of soil-transmitted helminth infections on overall health.
This brief summary highlights but a few notable video-articles that will be released in August. We'll also feature articles that demonstrate how to express genes in the midbrain of the chick embryo, study neutrophil transmigration in parallel flow chambers lined with endothelial cells, and precisely measure proteolytic degradation of the extracellular matrix.

Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and mirrors to refract and direct light to optical sensors. The August 2012 issue of JoVE (Journal of Visualized Experiments) is marked by the third publication from the Ozcan Lab (University of California, Los Angeles) on their lens-free "on-chip" microscopy platform, which they have pioneered.

Traditional microscopy requires lens objectives to magnify specimens, and can involve numerous optical components like additional objectives, filters, and ...

Research

JoVE Journal

Biology

2012: A Year In Review

Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).
At the start of 2012, JoVE reached a major milestone by publishing our 1500th article. In this publication, in JoVE Bioengineering, Yu et al. described a method for analyzing the free radical composition of cigarette smoke. This method simulated cigarette puffing with a single-port smoking device, and combined it with electron spin resonance (ESR) spectroscopy to measure the degree to which antioxidant compounds can scavenge free radicals in cigarette smoke. A possible outcome of this research is the creation of less harmful cigarettes.
JoVE Bioengineering also contained the most-viewed article of 2012. Owczarczak et al. described a method for hacking a standard inkjet printer, loading ink cartridges with a cell-suspension "bioink," and printing lines of live cells onto glass slides. The thermal inkjet printing process creates temporary pores in the cell membrane, visualized by the incorporation of fluorescently labeled actin monomers. This concept, called "bioprinting," has many potential applications in cell and tissue engineering.
Also in 2012, JoVE Bioengineering featured a protocol by Hsia et al. for isolating and purifying spider silk protein, spinning it into fibers, and assessing the fiber strength. This protocol for laboratory-scale production of spider silk, which is stronger than tensile steel, can potentially be extended to large-scale manufacturing.
In JoVE Clinical and Translational Medicine, we featured a number of articles related to stroke, one of which. In one article, M&ouml;bius-Winkler et al. demonstrated placement of the WATCHMAN left atrial appendage occlusion device from Boston Scientific. This device is designed to prevent strokes by trapping clots within the left atrial appendage of the heart before they exit. Our authors demonstrated how to insert the guide wire through the femoral artery into the heart, advance the device into the left atrium, and deploy the device in the left atrial appendage.
Another article focused on stroke, Hamel et al. described the use of a driving simulator to study how patients compensate for visual field defects following stroke. By analyzing the compensatory gaze behavior of patients as they navigated through virtual driving courses with varying degrees of complexity, our authors see great potential for the use of driving simulators in stroke rehabilitation.
In JoVE Immunology and Infection, we published an article by Keyel et al. describing the real-time kinetics of immune cell responses to bacterial toxins using live cell microscopy. Combined with high-speed 3D confocal microscopy, this technique can also visualize the cellular repair response.
Also in JoVE Immunology and Infection, we featured an article by Yap et al. describing methods for diagnosing helminth infections in children. Helminths are parasitic worms that can infect the intestinal tract of humans and animals, and may be distinguished based on their morphology. The same article demonstrated how to measure the impact of helminth infections on physical fitness in children, which is a measure of overall health.
In JoVE Neuroscience, we featured a method by Hoffmann et al., who used tiny headphones to alter the auditory feedback in songbirds, and analyzed the computational and neurophysiological basis of vocal learning in birds when they adjust their singing in response to altered acoustic signals.
Also in JoVE Neuroscience, we moved from birds to bees with a method that assesses associative and non-associative tactile learning in honeybees. This method, by Mujagi&#263; et al., allowed bees to scan different metal surfaces with their antennae, then conditioned them to expect sugar water when their antennae touched particular surfaces, and analyzed the corresponding changes in antennal movement.
In 2012, we launched a brand-new section: JoVE Applied Physics, which features articles on subjects ranging from plasma physics to material science. The Applied Physics section also contained JoVE's 2000th video article, filmed in a synchrotron radiation facility, where Borisenko et al. showed how to determine the electronic structure of complex materials using angle-resolved photoemission spectroscopy.
In the JoVE General section, we published an article by Artioli et al. demonstrating the use of a portable gas analyzer to measure oxygen consumption during complex exercise - such as judo. By analyzing oxygen consumption and collecting blood for measuring plasma lactate concentration, this method can determine the relative contributions of different energy systems to specific aspects of complex exercise.
Finally, JoVE followed Dolhi et al. to a permanently ice-covered saline lake in Antarctica. There, scientists drilled through the ice to study single-celled microorganisms called protists that live in the harsh conditions of Antarctica.
This Year in Review was just a brief glimpse of a few of over 600 video articles that JoVE offered in 2012. Browse the JoVE archives to see thousands of other videos, and stay tuned for what's coming up this year in JoVE: The Journal of Visualized Experiments.

Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).

 
 
Here's a look at some of the milestones and highlights of the year 2012 in Journal of Visualized Experiments (JoVE).
At the start of 2012, JoVE ...